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With buildings across the East Rockaway School District in need of repairs and upgrades, officials have scheduled a facilities tour at the high school on Saturday at 11 a.m.
The walk-through is intended to give residents a better sense of the district's facility needs as administrators mull a possible $30.4 million bond to cover needed projects. The Facilities Committee, comprising Board of Education trustees, teachers, parents, administrators and district architect John Grill, will outline the proposed renovations.
Superintendent Lisa Ruiz said the committee did an "outstanding job of narrowing down the scope of the work," adding, "Engagement with our residents is one of our top priorities during this time."
Voters approved an $18.7 million bond on March 18, 2005, which will be paid off in 2020. If district officials were to put another bond to a vote, payments would not be made until after the current one is paid off, said Jacqueline Scrio, the district's assistant superintendent of finance and operations.
"There is no definite decision on the bond," Scrio said. "It's been many years since we've had a bond or we've done any large improvements to the district."
The committee met six times between February and June 2018 and compiled a list of needs at the high school and Rhame Avenue and Centre Avenue elementary schools. They included air conditioning and ventilation installation, masonry improvements and classroom upgrades. Scrio said the consumer science classrooms are outdated, and district officials narrowed their focus after Grill provided a cost analysis for each project.
Scrio added that officials hoped to decide on whether the district will propose another bond by June. She noted that it is difficult to determine how much the bond would impact homeowners because a county-wide reassessment is ongoing, but she estimated that the bond would cost the average home about $104 per year.
A major concern officials hope to address is the rapid erosion of the playing fields at the high school as the shoreline of Mill River creeps closer to the grass with each passing storm. "There's 42 inches between the shoreline of Mill River and the back of the bleachers because there's no bulkhead," Scrio said. "We're losing our fields slowly but surely."
The district is awaiting approval of a major project that falls under Living with the Bay — a state initiative undertaken after Hurricane Sandy and funded by the Governor's Office of Storm Recovery to improve storm resiliency in communities along Mill River, which runs from Hempstead Lake south to Hewlett Bay.
The district reached an agreement with GOSR to install a Greenway Project, which is to include a path for walking and biking from Hempstead Lake State Park through the Village of East Rockaway and behind the high school, on district property. As part of the agreement, GOSR is to fund a $1.9 million bulkhead, which district officials said would protect the fields at no additional cost to local taxpayers.
"It benefits GOSR," Scrio said, "because they'll get access to the waterfront, and it will benefit the district because in order to do the Greenway, they'll add a bulkhead."
In August, school officials presented the projects they deemed necessary to residents at a meeting, but did not move forward because they were waiting on a timeline from Living with the Bay. Scrio said that officials hoped that GOSR representatives would meet with them and residents to provide a clear timeline for the project, but have not heard back. The LWTB plans have been under discussion since 2016.
Emily Thompson, the acting general counsel and chief external affairs officer for GOSR, said the next meetings were scheduled for May.
"GOSR is committed to a collaborative and structured design and review process with the community and school administrators and looks forward to its next engagement in early May," she said. "The resiliency improvements at East Rockaway High School will protect the school and residents from flooding, defend the shoreline from erosion and foster community engagement along the river. GOSR looks forward to presenting the specific improvements to the community in the coming months."
Officials also said they hoped to install a stronger drainage system beneath the fields and to raise them to avoid further flood damage. Inside the schools, pipes dating back to the 1930s are eroding and need to be replaced, as do many of the floors.
Another large project would be the addition of a hall that would connect the high school's technology building to the main building. To merge the now separate areas, workers would have to demolish a garage and build a driveway between the technology building and the athletic fields. They would also install a 15-foot-tall chain-link fence to separate the fields from the driveway leading to the back parking lot.
In January, the Board of Education hosted a facilities forum, at which trustees updated residents on the status of the Greenway Project and discussed the possibility of bonding for other needs. Scrio said she hoped that GOSR would have the bulkhead in place by the summer of 2020, and that the fields would be worked on that fall.
She said that future school tours would likely be announced. "Showing people this, seeing it, visualizing it, really makes the difference," she said.
Ruiz encouraged residents to take the tour. "I urge everyone to complete the facilities survey posted on our website and sign up to attend our upcoming facilities tour," she said.
Visit eastrockawayschools.org to complete the survey.
Melissa Koenig contributed to this story.


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text:
|Kreg Leymaster
The central theme of Dr. Leymaster's research program is the evaluation, development, and use of dam and sire breeds of sheep in terminal crossbreeding systems. Dam breeds are studied by crossing with Romanov to capture the documented superiority of Romanov crossbred ewes for fertility, length of seasonal fertility, prolificacy, lamb survival, and ewe longevity. Wool (Dorset and Rambouillet) and hair (Dorper and Katahdin) breeds are evaluated for their ability to complement Romanov genetics in intensive and low-input production systems and in fall and spring breeding seasons. In the intensive production system (barn lambing), ewes are limited to naturally rearing two lambs with additional lambs reared artificially. Ewes in the low-input production system (pasture lambing) are completely responsible for rearing of all lambs. The long-term goal is to create an easy-care dam line of hair sheep that can rear triplets on pasture with no labor or supplemental feed. Research with dam breeds is complemented by research with terminal sire breeds. A terminal sire composite population was formed by mating


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Even today, buffer overflows are a threat to computer-system security. Over the last few years, vendors of operating systems have set up their products with different strategies to prevent buffer overflow attacks. Address space layout randomization (ASLR) and non-executable stacks are two such improvements that try to prevent attackers from using vulnerabilities based on buffer overflows for a successful intrusion.
In addition, compiler vendors added techniques against buffer overflows, especially stack overflows, to their products. All major vendors of C and C++ compilers have added detectors to their compilers that can sense stack smashing the willful use of stack overflows to gain control of a system before any harm can be done. They cannot prevent the stack from being smashed, but they can detect the smashing and avoid executing the malware payload. Such "buffer protectors" include Stack Smashing Protector (SSP) for GNU's gcc, ProPolice for IBM's XLC, and Buffer Security Check for Microsoft's Visual compilers (option /GS). In this article, I examine in detail how stack overflows occur, how stack smashing attacks work, and how they can be detected. I also examine the implementation of the GNU SSP and show the methods it employs to thwart these attacks.
Understanding the Problem
Before discussing a solution, you have to understand the problem : What happens in the case of a stack overflow and why is it so dangerous?
Whenever a program calls a subroutine by a CPU instruction
CALL or
BRANCH, it saves the current instruction pointer (IP) onto the stack. It marks its position before it branches, so it knows where to return after termination of the subroutine. This saved address on the stack is called a return address.
A high-level programming language like C or C++ also puts local variables of the subroutine on top of the stack (Figure 1). Thus, the subroutine gets its own memory area on the stack where it can store its own data. This principle is also the key of recursive routine calls because every new call to the subroutine gets its own return address and its own local variables on the stack.
Figure 1: Stack layout.
Upon termination of the subroutine, high-level languages first clean up the stack by removing all local variables. After that, the stack pointer (SP) again points to the saved return address. At a
RET or
RETURN instruction, the processor reads the return address from stack, jumps back to the former IP position, and continues the original program flow.
This flexible layout allows theoretically infinite levels of recursion, but several problems are also inherent. If a program does not check the boundaries of local variables at writing, it could write beyond the intended space of these variables. In the best case (from an operating system's perspective), this will overwrite other variables. In the worst case, it will overwrite the return address. Why? On most systems, including state-of-the-art systems, the stack and heap of a program are growing toward the same point in memory. Where the heap grows from lower to higher addresses, the stack grows from higher to lower addresses (see Figure 2). In other words: The stack stands upside down.
Figure 2: The memory layout of a process.
Even if the stack grows from higher to lower addresses, the local variables on the stack work the "normal" way: They grow from lower to higher addresses. Any string and any array store their values of lower indexes at lower addresses and higher ones at higher addresses.
Let's assume a program writes data to a local string variable, by
strcpy() for example.
strcpy() does not check for any boundary. It copies data to the destination variable until it reads a null byte from the source variable. Thus, it is able to write more bytes onto the stack than there is space dedicated to the provided string variable. As a result, it might overwrite other data in higher addresses in memory and therefore below the destination variable on "lower addresses" on the stack (see Figure 3). If the copy process is not stopped, sooner or later, the program will overwrite the return address, too. The return address will be destroyed, or more precisely, given a new value.
Figure 3: A stack overflow.
At termination of the subroutine, when the program retrieves the return address, that address has changed. Under normal circumstances, the program flow jumps to an unpredictable address. Chances are very good that the jump leads into a segment outside the program's own memory. The system detects a segmentation violation and kills the process with signal number 11 (
SIGSEGV). The program crashes and leaves a
core file.
So far, so good. But what happens if the target of the changed return address is not outside the program's own memory? In this case, the jump might succeed and execution will go on. If there is valid machine code, it will be executed. If not, the processor will create an illegal instruction exception.
Footprint of a Stack Overflow Attack
Attackers change the return address via a buffer overflow not to an unpredictable location, but to a specific address. In other words: They can control where the jump goes. A classic attack includes a so-called "payload" (also called a "cuckoo's egg") in the overflowing data, which consists of three parts:
- An NOP sled
- A shell code
- A new return address
The NOP sled consists of a repeated processor instruction, called
NOP (no operation). This instruction does nothing but increase the IP by one. In other words, it's a do-nothing filler byte.
The shell code is a little program written by the attacker in assembly language and machine code, respectively. Normally, it utilizes system calls to gain a root shell. This is why it is called "shell" code.
Last but not least, the new return address is provided, which points somewhere into the NOP sled. If the subroutine terminates, the program flow will jump into the NOP sled and execute the NOPs until it reaches the shell code (see Figure 4). The shell code opens a root shell and the attacker gains control over the system.
Figure 4: A stack overflow attack.
The NOP sled is simply a way to avoid knowing the precise address of the shell code on the stack. Systems differ, and calculating the exact return address is an art of its own. It is easier to jump somewhere into the middle of the NOP sled. Stack layout will differ from system to system and from program execution to execution. It won't matter if the payload lays a bit higher or lower on the stack. As long as the jump goes inside the NOP sled, the exploit will work. For the same reason, the return address is not simply one single value. It is a concatenation of repetitions of the intended return address. One of these repeated addresses will overwrite the original return address on the stack.
Attacks with shell code in the stack segment don't work on contemporary systems anymore. Features like non-executable stacks, provided by the hardware or operating system, prevent execution of injected code on the stack. But the threat is not yet removed entirely. There are other tricks that an attacker can use. The "Return to libc" is one such trick. Such attacks also rely on stack overflows, but they are a bit more complicated. For a stack smashing protector, it makes no difference whether it is a classic or modern attack. So I'll concentrate on classic attacks for the sake of simplicity.
Protecting Canaries
The classic stack overflow attack and most of its modern successors are organized in two steps. The first step is a preparation stage in which the payload overwrites the return address. After this, the attacker has to wait while the "normal" algorithm of the vulnerable subroutine goes on. Only when the subroutine terminates will the prepared return address force the CPU to start the real attack with its harmful impact.
Thus, there is a temporal and operational gap between the preparation and the implicit invocation of the injected code. For a protection mechanism, this gap is predestined to get a closer look on the stack before returning from the subroutine. In no case can an attacker change the code of the subroutine itself. So the subroutine's code always stays intact.
The aforementioned stack smashing protectors of contemporary compilers use exactly these two findings the gap and the untouched code of the subroutine in this gap. They add additional code to the subroutine as a prologue and an epilogue that detect stack overflows.
A typical subroutine of a language like C or C++ consists of four parts at the machine level:
- Initialization: the preparation of space on the stack for local variables.
- Subroutine body: the subroutine's implemented algorithm.
- Clean-up: removing local variables from the stack.
- Return: jump back to the original address before the branch.
Stack smashing protectors (SSP) add two additional parts to this schema. A special prologue, which is placed before the initialization, and an epilogue, which fits between clean-up and return. This leads to the following new layout of a subroutine:
- SSP's prolog
- Initialization
- Clean-up
- SSP's epilog
- Return
The idea is to add a barrier on the stack between the return address and the local variables. In case of a buffer overflow on the stack, the barrier will be crossed and destroyed. SSP's epilog will detect this invalid barrier. The barrier is simply an integer value and is called a "canary" in SSP's own wording. Before the return address is destroyed, the canary will be destroyed (see Figure 5).
Figure 5: Destroyed canary indicating stack smashing.
The destroyed canary indicates stack smashing. A simple comparison with the SSP's epilog shows the changed canary and uncovers the overflow and a potential attack, respectively. All this is detected before any branch based upon whether a changed return address might occur. The program doesn't lose control and it can stop its process in the SSP epilog before the return step and before any potential damage is done.


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"To travelers around the world, Edward Hasbrouck is The Practical Nomad, the go-to authority on international travel, and an expert on airfares and how to get the best deals on the Internet", says Business Week. Hasbrouck is the world's best-known authority on around-the-world travel: consumer advocate, investigative journalist, speaker, blogger, author of the acclaimed Practical Nomad series of travel how-to and advice books, and consultant on travel-related human rights and civil liberties issues with the Identity Project.
Backpackers and business travellers alike -- people who are on their own without a tour guide -- rely on Edward Hasbrouck's expertise and advice about independent travel:
"I write and speak about travel as an aspect of life: something we do, not something we buy. There is an industry of companies and people who sell services to travellers, but travel is an activity, not an industry; an experience, not a product. We may pay for transportation and for places to sleep, just as we may pay for schooling, but we can't buy experience any more than we can buy enlightenment -- even if, in the best of worlds, travel sometimes brings us both. I abhor the commodification of travel, and I write and speak about how my readers and listeners can avoid it."
"A journalist and travel expert, Mr. Hasbrouck brings to the table in-depth knowledge of the travel industry," says the Wall Street Journal."That rare person, the expert who's willing to tell all. Edward Hasbrouck knows as much about cheap airline tickets as anyone," according to the Whole Internet Catalog. Amazon.com recommends, "If you've decided to put your money where your fantasy is, and save for the trip of trips, Edward Hasbrouck is your travel guru". Tripod.com calls him, "A nationally renowned expert [on how to] get the best of the budget deals without getting burned."
Hasbrouck's blog has been recognized and recommended by reviewers from the New York Times and the Wall Street Journal to the Times and the Telegraph (UK) as one of the leading travel blogs. The Practical Nomad: How to Travel Around the World -- in print for more than 15 years, and now in its 5th edition -- has been hailed by reviewers and readers alike as the bible of independent international travel how-tos.
Hasbrouck has been featured in a BBC-TV travel documentary, spoken at National Geographic Society headquarters in Washington, and been quoted and interviewed as a travel expert by "The Savvy Traveler" and "Marketplace" on NPR, the "TODAY Show" on NBC-TV, "Computer Chronicles" on PBS, CNN, C-SPAN, CNBC, TechTV, CBC Radio, Pacifica Radio, Voice of America, on the front pages of the the New York Times and the Washington Post, and in the Toronto Star, London Sunday Times, Guardian (U.K.), Le Monde (France), Heise (Germany), Zeit (Germany), Los Angeles Times, Christian Science Monitor, USA Today, Chicago Tribune, Miami Herald, AP, UPI, Reuters, Newsweek, Business Week, Wired News, Salon.com, CBS MarketWatch.com, FoxNews.com, TheStreet.com, Bankrate.com, Aviation Week, TravelAge West, Travel Weekly, Airline Financial News, World Airline News, Business Travel News, Kiplinger's Personal Finance, National Geographic Traveler, National Geographic Adventure, The New Yorker, The Intercept, and numerous other media outlets. In the San Francisco Bay Area, he's been seen on KGO, KRON, and KTVU television; heard on KPFA, KQED, KPOO, and KALW public radio; and quoted in the San Francisco Chronicle, San Jose Mercury News, Oakland Tribune, and Contra Costa Times.
The winner of a 2003 Lowell Thomas Travel Journalism award for investigative reporting from the Society of American Travel Writers Foundation for his in-depth reporting on the privacy of travel records, Hasbrouck has been in the forefront of efforts to protect the freedom to travel (as a human right recognized and guaranteed by international human rights law as well as as the First Amendment to the U.S Constitution), the privacy of travellers, and their right to control how their personal travel records are used by travel companies and government agencies. He has testified before the Dept. of Transportation Advisory Committee for Aviation Consumer Protection, the TSA, the DHS Data Privacy and Integrity Advisory Committee, state legislatures in California and other states, the Canadian House of Commons, and the European Parliament, and works with the Identity Project (part of the First Amendment Project). He has contributed articles on travel and privacy to Privacy Journal, Privacy International's Privacy and Human Rights yearbook, and Privacy Laws & Business International Report, and has participated in numerous U.S. and European Union regulatory proceedings. Hasbrouck has provided declarations as an expert witness on airline reservations technology, data, and business practices in successful public interest and consumer litigation and arbitration against government agencies, airlines, and other travel companies in both the USA and Canada.
Hasbrouck is a passionate, articulate, and dedicated defender of travellers' rights. His consumer and civil rights advocacy has earned him praise and respect from his readers, supporters, and allies, and the personal animus of both politicians and businesses he has criticized. Industry commentators and politicans have denounced him on the op-ed pages of The New York Times (as a "yuppie") and the Wall Street Journal (as a "privocrat"), while internal e-mail messages between high-level TSA and DHS officials, later released in response to requests under the Freedom Of Information Act, call him everything from "a well-known Privacy Advocate" to "crazy as a loon" and "impossible to deal with".
In 2003, Hasbrouck broke the news on his Web site and in his e-mail newsletter that jetBlue Airways had provided 5 million reservation records to contractors for the USA government's CAPPS-II and Total Information Awareness programs, a week before the story hit the front pages of newspapers across the country and around the world. More recently, he's played a leading role in exposing the threats to travellers' rights posed by RFID chips in passports, the new long form application for a US passport, and government surveillance and control of travellers' movements. In 2011, he sued the US Department of Homeland Security under the Privacy Act and the Freedom of Information Act to obtain the government's files of his travel history, airline reservations, etc. from the DHS "Automated Targeting System". (In response, the DHS retroactively exempted these records from the U.S. Privacy Act.) In 2016, hackers publicly confirmed severe security and privacy vulnerabilities in the Web sites that give access to airline reservations that Hasbrouck had been the first to write about 15 years earlier.The Washington Post calls Hasbrouck "an expert on travel data privacy", and his groundbreaking investigative reporting and research on privacy policy and technology issues for travellers has been cited everywhere from Scientific American, Inside ID, and Reason to Surveillance & Society and the Columbia Science and Technology Law Review.
Hasbrouck has spoken at:
Hasbrouck's travel how-to and advice books include a consumer guide to using the Internet for travel planning and purchasing, The Practical Nomad Guide to the Online Travel Marketplace (2001), and the definitive guide to independent international travel, The Practical Nomad: How to Travel Around the World (5th edition, 2011).
Hasbrouck has gone around the world three times himself, once in each of the last three decades. While updating the most recent edition of The Practical Nomad: How to Travel Around the World, he spent 13 months on an 80,000-mile trip through 28 countries on 6 continents. Hasbrouck has visited all 50 states of the USA, 8 Canadian provinces, and more than 60 other countries, travelling by foot, bicycle, train, bus, boat, car, taxi, rickshaw, donkey and pony cart, and on more than 60 different airlines. In recent years, he has bicycled thousands of miles across the USA, Canada, Europe, and Argentina.
Hasbrouck has worked with thousands of independent travellers to help them plan and organize their trips. Long active in peace and human rights organizations, Hasbrouck is an evangelist for travel as a positive force for global change. In addition to his travel books and articles for travel magazines, Hasbrouck has written for a variety of publications on peace, justice, and human rights, including Peacework, The Nonviolent Activist, Antiwar.com, and Privacy Journal.
For more than 15 years, Hasbrouck worked for offline and Internet travel agencies specializing in independent around-the-world travel, with clients including individual travellers and groups as well as travel technology and consulting organizations. He also remains a paid affiliate of Airtreks.com, where he was the staff "Travel Guru" from 1998 to 2006 before he shifted his focus to full-time freelance writing, Web publishing, and consulting.
Hasbrouck is a member of numerous professional associations and peace and human rights groups, including the National Writers Union (Co-Chair, Book Division), the Bay Area Travel Writers (former member, Board of Directors), the National Lawyers Guild (non-lawyer legal worker member), the War Resisters League, Hostelling International (life member and member of the Honorary Committee for HI-USA's 75th Anniversary Campaign for Hostelling), the National Association of Railroad Passengers, and the San Francisco Bicycle Coalition.
As Co-Chair since 2009 of the Book Division of the National Writers Union, Hasbrouck has been active in the NWU's advocacy, legislative, and litigation work for writers in the USA and abroad, He has testified for the NWU to Congress and the U.S. Copyright Office on issues related to writers' rights and economic interests, and represented the NWU in meetings with Congress, foreign governments, international organizations, and alliances of writers' groups including the International Authors Forum (IAF) and the International Federation of Journalists (IFJ), with which the NWU is affiliated. From 2016 through 2019, he served on the Board of Directors of the International Federation of Reproduction Rights Organizations (IFRRO), holding the sole seat on that board reserved for an elected representative of writers worldwide.
The Practical Nomad series of books by Edward Hasbrouck is published by Avalon Travel Publishing, a division of the Hachette Book Group. Other Avalon Travel imprints include Moon Handbooks, Rick Steves' Guides, Living Abroad In..., Road Trip USA, The Peoples Guide to Mexico, and Foghorn Outdoors.
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Copyright © 1991-2020 Edward Hasbrouck, except as noted. Use of any information obtained from this site for the purpose of sending unsolicited bulk e-mail is expressly forbidden, and is a violation of your license to use this copyrighted material.


text:
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text:
Netflix Gets Wells Notice Over CEO Hastings's Facebook Post
Netflix and CEO Reed Hastings both received Wells Notices from the SEC, according to a filing this afternoon related to something Hastings wrote on Facebook back in June.
Back in July, Hastings wrote on his Facebook page that Netflix users had streamed more than 1 billion hours of video in June. The SEC is apparently looking into whether or not that violates fair-disclosure rules.


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In a cruel new world, it only seems fitting that Peter and Olivia would reunite with their daughter only to have her tragically taken away from them too soon.
On last Friday's Fringe, the Observers mercilessly killed the youngest Bishop family member, sending out shock waves among the Fringe fandom. How could Etta (Georgina Haig), who we came to love during such a short period of time, be taken away so soon? This is just a taste of the danger Peter (Joshua Jackson), Olivia (Anna Torv), Walter (John Noble) and Astrid (Jasika Nicole) will continue to face in the Observer-occupied future.
Mega Buzz: Which familiar face is returning to Fringe?
"Etta's death is horrendous because, particularly this family — Peter, Olivia, Walter and Astrid — have had so much loss," Noble tells TVGuide.com. "The whole series is predicated on loss. The loss of Peter is what caused all this. To see them united as a family and getting back together, because Peter and Olivia had been alienated too, the death of their little girl, just when we've gotten to really love her, it's tragic."
The remaining Fringe team will be bonded through this tragedy because they're all they have left against the Observers. "It adds a whole other layer of despair to this really dire situation that they're in," Nicole says, explaining that they've essentially lost their inside person who actually lived through the Observer occupation. "In a way she's kind of like this bright light in this awful, desperate place that they find themselves in. The one thing that they had that was kind of keeping them afloat is not there anymore."
For Olivia, it's a chance to handle the loss of her daughter in a different way this time around. "The next episode for Olivia is where she makes a decision to mourn differently this time and to handle her grief differently, and to try and find a little bit more of a shared thing with Peter through it, instead of them separating like they did," Torv says. "For Olivia, you see this moment of, 'Oh sh--, if this has happened again, then I'm not going to make the same mistakes again.' They pick themselves up as best they can."
But based on the promo for this week's episode, Peter is on a dangerous path for vengeance, which may cause a bit of a divide between him and Olivia. "They each have to cope with it in their own way," Jackson says. "The choices that they each make, particularly the choices that Peter makes, are not very constructive. So it's not necessarily that it's driven a wedge between the two of them, but they're not really available for each other in a way that they would hope to be dealing with a tragedy."
Eventually, the loss and pain will be too much for someone on the team — though whether that's actually Peter remains to be seen. "At some point in the future, it's a breaking point for one of the characters," Nicole says. "It's just too hard for them accept that. Her death was something that needed to happen. They've gone through a lot of really difficult situations, but this is the one situation that they were completely unprepared for. It's really hard to watch them struggle through the ensuing episodes."
Fringe's final season is a 13-episode feature film
Jackson agrees that Etta's death was a necessary evil in Fringe's journey to the end. "If the idea that [executive producer J.H.] Wyman had for the show this year that it was a play in three acts, this finishes the first act of the play," he says. "Obviously the death of their child and Walter's grandchild changes everything, changes the stakes of everything and it changes the motivation for everything going forward for all of the characters. Each one of them, over the course of the five episodes that follow, are just dealing purely with the fallout of that in their various ways. Then, at the end of that second act, they shift paths again to get us into our homestretch."
Check out more from the cast of Fringe on how Etta's death will change everything moving forward:
What did you think of Etta's death on Fringe?
Fringe airs Fridays at 9/8c on Fox.


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We can integrate Infusionsoft with a number of other websites. Synchronize, organize, connect, and free your data.
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text:
Overview The Invitrogen T-REx System is a tetracycline-regulated mammalian expression system that uses regulatory elements from the E. coli Tn10-encoded tetracycline (Tet) resistance operon (Hillen and Berens, 1994; Hillen et al., 1983). Tetracycline regulation in the T-REx System is based on the binding of tetracycline to the Tet repressor and derepression of the promoter controlling expression of the gene of interest (Yao et al., 1998). The major components of the T-REx System include:
- An inducible expression plasmid for expression of your gene of interest under the control of the strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites
- A regulatory plasmid, pcDNA6/TR™, which encodes the Tet repressor (TetR) under the control of the human CMV promoter Tetracycline for inducing expression
- A control expression plasmid containing the lacZ gene, which when cotransfected with pcDNA6/TR™, expresses β-galactosidase upon induction with tetracycline.
For specific information on the inducible expression vector and the corresponding positive control vector containing the lacZ gene, please refer to the manual for the inducible expression vector you are using.
Description of the T-REx System
In the T-REx System, expression of your gene of interest is repressed in the absence of tetracycline and induced in the presence of tetracycline (Yao et al., 1998). Unlike other tetracycline-regulated systems which use hybrid regulatory molecules and viral transactivation domains (Gossen and Bujard, 1992), the T-REx System uses only regulatory elements from the native Tet operon (Yao et al., 1998). Tetracycline-regulated gene expression in the T-REx System more closely resembles the regulation of the native bacterial tet operon (Hillen and Berens, 1994; Hillen et al., 1983) and avoids the potentially toxic effects of viral transactivation domains observed in some mammalian cell lines.
The major component of the T-REx System is the inducible expression plasmid. Expression of your gene of interest from the inducible expression vector is controlled by the strong CMV promoter (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987) into which 2 copies of the tet operator 2 (TetO2) sequence have been inserted in tandem. The TetO2 sequences consist of 2 copies of the 19 nucleotide sequence, 5´-TCCCTATCAGTGATAGAGA-3´ separated by a 2 base pair spacer (Hillen and Berens, 1994; Hillen et al., 1983). Each 19 nucleotide TetO2 sequence serves as the binding site for 2 molecules of the Tet repressor. For more information about the Tet operator sequences and the specific features of each inducible expression vector, please refer to the manual for the vector you are using.
The second major component of the T-REx System is the pcDNA6/TR regulatory vector which expresses high levels of the TetR gene (Postle et al., 1984) under the control of the human CMV promoter. Both T-REx vectors can be introduced into mammalian host cells by standard transfection methods.
Mechanism of repression
In the absence of tetracycline, the Tet repressor forms a homodimer that binds with extremely high affinity to each TetO2 sequence in the promoter of the inducible expression vector (Hillen and Berens, 1994). The 2 TetO2 sites in the promoter of the inducible expression vector serve as binding sites for 4 molecules (or 2 homodimers) of the Tet repressor. The affinity of the Tet repressor for the tet operator is KB = 2 x 1011 M-1 (as measured under physiological conditions), where KB is the binding constant (Hillen and Berens, 1994). Binding of the Tet repressor homodimers to the TetO2 sequences represses transcription of your gene of interest. Upon addition, tetracycline binds with high affinity to each Tet repressor homodimer in a 1:1 stoichiometry and causes a conformational change in the repressor that renders it unable to bind to the Tet operator. The association constant, KA, of tetracycline for the Tet repressor is 3 x 109 M-1 (Hillen and Berens, 1994). The Tet repressor: tetracycline complex then dissociates from the Tet operator and allows induction of transcription from the gene of interest.
Experimental outline
The gene of interest is cloned into the multiple cloning site of the inducible expression vector, and the resulting construct cotransfected with the regulatory plasmid, pcDNA6/TR™ into mammalian cells. After transfection, cells are treated with tetracycline to derepress the hybrid CMV/TetO2 promoter in the inducible expression vector and induce transcription of your gene of interest.
The positive control vector containing the lacZ gene can be transiently cotransfected into mammalian cells with pcDNA6/TR™ to demonstrate that the system is working properly in your cell line. Stable cell lines expressing Tet repressor from pcDNA6/TR™ can be established to serve as hosts for inducible expression vector-based constructs.
The T-REx System manual is supplied with the kits listed below. The Core System includes the inducible expression vector of choice, the regulatory vector, and primers for sequencing. The Complete System includes the Core System plus inducing and selection agents. See below for a detailed description of the contents of each T-REx Kit.
The T-REx Core System is shipped at room temperature. Store at -20°C.
The T-REx Complete System is shipped in 2 boxes. Store as described below:
- Box 1 contains vectors, primers, and blasticidin and is shipped at room temperature. Upon receipt, remove the vectors and primers and store at -20°C. Blasticidin powder should be stored at +4°C.
- Box 2 contains tetracycline and Zeocin and is shipped on blue ice. Store at +4°C. For long-term storage (> 6 months), store at -20°C. Store the tetracycline and Zeocin protected from exposure to light.
Kit contents
Both the T-REx Complete and the T-REx Core Systems include the following regulatory vector and sequencing primers. Store at -20°C.
20 µg, lyophilized in TE, pH 8.0
Regulatory vector that expresses the tetracycline (Tet) repressor
CMV Forward Primer
2 µg (306 pmoles), lyophilized in TE, pH 8.0
BGH Reverse Primer
2 µg (358 pmoles), lyophilized in TE, pH 8.0
Inducible expression vector
Each T-REx Complete and Core System also includes one of the following inducible expression vectors and a corresponding positive control vector containing the lacZ gene. Please refer to the vector manual for specific information pertaining to each inducible expression vector. Store at -20°C.
Amount supplied
5 g, powder
Inducing agent
1 g
Selection agent for inducible expression plasmid
50 mg, powder
Selection agent for pcDNA6/TR™ plasmid
T-REx cell lines
For your convenience, Invitrogen has available three mammalian cells lines that stably express the Tet repressor. T-REx-293 cells and T-REx-HeLa cells express the Tet repressor from pcDNA6/TR™ and should be maintained in medium containing blasticidin. T-REx-U2OS cells express the Tet repressor from pCEP4/tetR as described in Yao et al., 1998, and should be maintained in medium containing hygromycin. Please note that the pCEP4/tetR plasmid in the T-REx-U2OS cells is episomally-maintained, but is stable under hygromycin selection. For more information, please go to www.thermofisher.com or call Technical Service.
The following section contains guidelines for maintaining and propagating the pcDNA6/TR regulatory vector.
General molecular biology techniques
For assistance with E. coli transformations, restriction enzyme analysis, purification of single-stranded DNA, DNA sequencing, and DNA biochemistry, please refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).
E. coli strain
Many E. coli strains are suitable for the propagation of the pcDNA6/TR vector including TOP10F´ (Catalog no. C615-00), DH5αF´, and INVαF´ (Catalog no. C658?00). We recommend that you propagate the pcDNA6/TR vector in E. coli strains that are recombination deficient (recA) and endonuclease A deficient (endA).
For your convenience, TOP10F´ E. coli are available as chemically competent or electrocompetent cells from Invitrogen.
Catalog no.
One Shot TOP10F´ (chemically competent cells)
21 x 50 µl
Electrocomp TOP10F´ (electrocompetent cells)
5 x 80 µl
Max Efficiency DH10B Chemically Competent Cells
5 x 0.2 ml
Transformation method
You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.
Maintenance of plasmids
The pcDNA6/TR™ vector contains the ampicillin and blasticidin resistance genes to allow selection of the plasmid using ampicillin or blasticidin. To propagate and maintain the pcDNA6/TR plasmid, we recommend using the following procedure:
- Resuspend the vector in 20 µl sterile water to prepare a 1 µg/µl stock solution. Store the stock solution at -20°C.
- Use the stock solution to transform a recA, endA E. coli strain like TOP10F´, INVαF´, or equivalent.
- Select transformants on LB agar plates containing 50 to 100 µg/ml ampicillin or 50 µg/ml blasticidin in Low Salt LB.
- Prepare a glycerol stock of each plasmid for long-term storage (see protocol below).
Selection in E. coli
To facilitate selection of blasticidin-resistant E. coli, the salt concentration of the medium must remain low (< 90 mM) and the pH must be 7.0. Prepare Low Salt LB broth and plates using the recipe. Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug unless a higher concentration of blasticidin is used.
Preparing a glycerol stock
Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at -20°C.
- Streak the original colony out on an LB plate containing 50 µg/ml ampicillin or 100 µg/ml blasticidin in Low Salt LB. Incubate the plate at 37°C overnight.
- Isolate a single colony and inoculate into 1-2 ml of LB containing 50 µg/ml ampicillin or 50 µg/ml blasticidin in Low Salt LB.
- Grow the culture to mid-log phase (OD600 = 0.5-0.7).
- Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.
- Store at -80°C.
Plasmid preparation
Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipids decreasing transfection efficiency. We recommend isolating DNA using the S.N.A.P. MiniPrep Kit (10-15 µg DNA, Catalog no. K1900-01), the S.N.A.P. MidiPrep Kit (10-200 µg DNA, Catalog no. K1910-01) or CsCl gradient centrifugation.
Once you have obtained clean plasmid preparations of your inducible expression plasmid and pcDNA6/TR™, you are ready to cotransfect the two plasmids into the mammalian cell line of choice. We recommend that you include the positive control vector and a mock transfection (negative control) to evaluate your results. For more information about the positive control vector, please refer to the manual for the inducible expression vector you are using.
Methods of transfection
For established cell lines (e.g. HeLa, COS-1), please consult original references or the supplier of your cell line for the optimal method of transfection. We recommend that you follow exactly the protocol for your cell line. Pay particular attention to medium requirements, when to pass the cells, and at what dilution to split the cells. Further information is provided in Current Protocols in Molecular Biology (Ausubel et al., 1994).
Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989) and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). Invitrogen offers the Calcium Phosphate Transfection Kit for mammalian cell transfection and Lipofectamine 2000 Reagent to perform lipid-mediated transfection.
Tetracycline-reduced serum
When culturing cells in medium containing fetal bovine serum (FBS), please note that many lots of FBS contain tetracycline as FBS is generally isolated from cows that have been fed a diet containing tetracycline. If you culture your cells in medium containing FBS that is not reduced in tetracycline, you may observe low basal expression of your gene of interest in the absence of tetracycline. We have cultured our mammalian cells in medium containing FBS that may not be reduced in tetracycline, and have observed undetectable to very low basal expression of ß-galactosidase from the positive control vector in the absence of additional tetracycline. If your gene of interest produces a toxic protein, you may wish to culture your cells in tetracycline-reduced FBS.
For more information, please contact our Gibco FBS Sales Representatives to find the right FBS for your cell culture needs
Tetracycline (MW = 444.4) is commonly used as a broad spectrum antibiotic and acts to inhibit translation by blocking polypeptide chain elongation in bacteria. In the T-REx System, tetracycline is used as an inducing agent to induce transcription of the gene of interest from the inducible expression vector. Tetracycline induces transcription by binding to the Tet repressor homodimer and causing the repressor to undergo a conformational change that renders it unable to bind to the Tet operator. The association constant of tetracycline to the Tet repressor is 3 x 109 M-1 (Takahashi et al., 1991). Please note that the concentrations of tetracycline used to induce gene expression in the T-REx System are generally not high enough to be toxic to mammalian cells.
- Tetracycline is light sensitive. Store the powdered drug at +4°C in the dark. Prepare medium containing tetracycline immediately before use.
- Tetracycline is toxic. Do not ingest or inhale the powder or solutions containing the drug.
- Wear gloves, a laboratory coat, and safety glasses or goggles when handling tetracycline and tetracycline-containing solutions.
Preparation of tetracycline
To prepare a stock solution from the tetracycline salt supplied with the Complete Kit:
1. Weigh out 10 mg of tetracycline and transfer to a sterile 15 ml conical polypropylene tube.
2. Resuspend 10 mg of tetracycline in 10 ml of water to produce a 1 mg/ml stock solution that is yellow in color.
Note: If you are using a different form of tetracycline (i.e. free base form), resuspend in 100% ethanol rather than water.
3. Store the stock solution at -20°C protected from exposure to light.
Because tetracycline-regulated expression in the T-REx System is based on a repression/derepression mechanism, the amount of Tet repressor that is expressed in the host cell line from pcDNA6/TR will determine the level of transcriptional repression of the Tet operator sequences in your inducible expression construct. Tet repressor levels should be sufficiently high to suitably repress basal level transcription. We have varied the ratio of pcDNA6/TR and inducible expression plasmid that we transiently cotransfect into mammalian cells to optimize repression and inducibility of the hybrid CMV/TetO2 promoter in the inducible expression plasmid. We recommend that you cotransfect your mammalian host cell line with a ratio of at least 6:1 (w/w) pcDNA6/TR:inducible expression plasmid DNA.
Cotransfection and induction with tetracycline
Guidelines are provided below to cotransfect your inducible expression construct (or the control plasmid) and pcDNA6/TR into your mammalian cell line and to induce expression of your protein of interest with tetracycline. Since every cell line is different and may require a different method of transfection, some empirical experimentation may be needed to determine the optimal conditions for inducible expression.
- Use cells that are approximately 60% confluent for transfection.
- Cotransfect the pcDNA6/TR plasmid and your inducible expression construct at a ratio of 6:1 (w:w) into the cell line of choice using your preferred method. Absolute amounts of plasmid will vary depending on the method of transfection and the cell line used.
- After transfection, add fresh medium and allow the cells to recover for 24 hours before induction.
- Remove medium and add fresh medium containing the appropriate concentration of tetracycline to the cells. In general, we recommend that you add tetracycline to a final concentration of 1 µg/ml (5 µl of a 1 mg/ml stock per 5 ml of medium) to the cells and incubate the cells for 24 hours at 37°C.
- Harvest the cells and assay for expression of your gene.
Optimization of expression
You may want to vary the concentration of tetracycline (0.1 to 1 µg/ml) and time of exposure to tetracycline (8 to 24 hours) to optimize or modulate expression for your cell line.
Other inducers
You may use doxycycline as an alternative inducing agent in the T-REx System. Doxycycline is similar to tetracycline in its mechanism of action, and exhibits similar dose response and induction characteristics as tetracycline in the T-REx System. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). Doxycycline may be obtained from Sigma-Aldrich (Catalog no. D9891).
Once you have established that your construct can be inducibly expressed, you may wish to establish a stable cell line that constitutively expresses the Tet repressor and inducibly expresses your gene of interest. We recommend that you first create a stable cell line that expresses only the Tet repressor, then use that cell line to create a second cell line that will express your gene of interest from the inducible expression plasmid. Alternatively, you can transfect with both plasmids (pcDNA6/TR and inducible expression vector) and dual-select with blasticidin and Zeocin to isolate a single stable cell line expressing both the Tet repressor and your gene of interest.
Three T-REx cell lines that stably express the Tet repressor are available from Invitrogen. If you wish to assay for tetracycline-inducible expression of your gene of interest in 293, HeLa, or U2OS cells, you may want to use one of the T-REx cell lines as the host to establish your double stable cell line. For more information, go to www.invitrogen.com or call Technical Service.
Reminder: When generating a stable cell line expressing the Tet repressor, you will want to select for clones that express the highest levels of Tet repressor to use as hosts for your inducible expression construct. Those clones that express the highest levels of Tet repressor should exhibit the most complete repression of basal transcription of your gene of interest.
Determination of antibiotic sensitivity
To successfully generate a stable cell line expressing the Tet repressor and your protein of interest, you need to determine the minimum concentration of each antibiotic (blasticidin and Zeocin) required to kill your untransfected host cell line. For each antibiotic, test a range of concentrations (see below) to ensure that you determine the minimum concentration necessary for your cell line. Use the protocol below to determine the minimal concentrations of Zeocin and blasticidin required to prevent growth of the parental cell line. Please refer to the Appendix, page 14 for instructions on how to prepare and store blasticidin. Instructions on how to prepare and store Zeocin can be found in the inducible expression vector manual.
- Plate or split a confluent plate so the cells will be approximately 25% confluent. For each antibiotic, prepare a set of 6-7 plates. Add the following concentrations of antibiotic to each plate in a set:
- For blasticidin selection, test 0, 1, 3, 5, 7.5, and 10 µg/ml blasticidin
- For Zeocin selection, test 0, 50, 125, 250, 500, 750, and 1000 µg/ml Zeocin
- Replenish the selective media every 3-4 days, and observe the percentage of surviving cells.
- Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth within 1-2 weeks after addition of the antibiotic.
Effect of Zeocin on sensitive and resistant cells
Zeocin's method of killing is quite different from other antibiotics including blasticidin, neomycin, and hygromycin. Cells do not round up and detach from the plate. Sensitive cells may exhibit the following morphological changes upon exposure to Zeocin:
- Vast increase in size (similar to the effects of cytomegalovirus infecting permissive cells)
- Abnormal cell shape
- Presence of large empty vesicles in the cytoplasm (breakdown of the endoplasmic reticulum and golgi apparatus, or other scaffolding proteins)
- Breakdown of plasma and nuclear membrane (appearance of many holes in these membranes)
- Eventually, these "cells" will completely break down and only "strings" of protein remain.
- Zeocin-resistant cells should continue to divide at regular intervals to form distinct colonies. There should not be any distinct morphological changes in Zeocin-resistant cells when compared to cells not under selection with Zeocin. For more information about Zeocin and its mechanism of action, please refer to the inducible expression vector manual.
Possible sites for linearization of pcDNA6/TR
To obtain stable transfectants, you may choose to linearize pcDNA6/TR before transfection. While linearizing the vector may not improve the efficiency of transfection, it increases the chances that the vector does not integrate in a way that disrupts the Tet repressor gene or other elements necessary for expression in mammalian cells. The table below lists unique sites that may be used to linearize your construct prior to transfection. Other restriction sites are possible.
Restriction site (bp)
Bst1107 I
AGS *, Fermentas, Takara
Sap I
New England Biolabs
BspLU11 I
Eam1105 I
Ampicillin gene
AGS *, Fermentas, Takara
Fsp I
Ampicillin gene
Selection of stable integrants
Once you have determined the appropriate antibiotic concentrations to use for selection, you can generate a stable cell line expressing pcDNA6/TR and your inducible expression construct.
- Transfect mammalian cells with pcDNA6/TR using the desired protocol. Remember to include a plate of untransfected cells as a negative control.
- 24 hours after transfection, wash the cells and add fresh medium to the cells.
- 48 hours after transfection, split the cells into fresh medium containing blasticidin at the pre-determined concentration required for your cell line. Split the cells such that the cells are no more than 25% confluent. If the cells are too dense, the antibiotic will not kill the cells. Antibiotics work best on actively dividing cells.
- Feed the cells with selective medium every 3-4 days until foci can be identified.
- Pick at least 20 foci and expand them to test for tetracycline-inducible gene expression by transiently transfecting with the positive control plasmid expressing beta-galactosidase. Screen for those clones that exhibit the lowest levels of basal transcription and the highest levels of β-galactosidase expression after addition of tetracycline.
- Once you have obtained a stable cell line expressing the Tet repressor, you can use this cell line to isolate a stable cell line expressing your gene of interest from the inducible expression plasmid. Repeat Steps 1 through 4, above, using your inducible expression vector construct and Zeocin to select foci. Remember to maintain your cells in medium containing blasticidin as well.
- Pick and expand at least 20 foci to test for tetracycline-regulated gene expression.
Dual selection of stable integrants
We recommend using the protocol above to generate double stable cell lines. However, if you wish to perform dual selection, you may cotransfect both pcDNA6/TR and your inducible expression construct into the cell line of choice. When cotransfecting your cell line, we recommend using a ratio of at least 6:1 (w/w) pcDNA6/TR DNA:inducible expression plasmid DNA to increase the chances that your double stable cell lines will express sufficient levels of Tet repressor. Following the steps listed above, add selective medium containing blasticidin and Zeocin at the concentrations determined previously. Screen at least 40 foci for tetracycline-regulated expression of your gene of interest using an appropriate assay for your protein (e.g. western blot, enzymatic assay).
Once you have isolated stable cell lines expressing both pcDNA6/TR™ and your inducible expression construct and have tested for tetracycline-inducible expression of your gene of interest, we recommend that you perform a time course of tetracycline induction to optimize expression of your protein of interest (e.g. 0, 8, 16, 24, 32 hours, etc.). Use the appropriate concentration of tetracycline for your cell line as previously determined. We have observed as much as 120-fold induction of β-galactosidase in a double stable cell line (T-REx 293 cells transfected with the positive control plasmid containing the lacZ gene) after 24 hours of treatment with 1 µg/ml tetracycline. Please note that your induction levels may vary depending on the nature of your gene of interest and the particular clone that you choose.
Map of pcDNA6/TR
pcDNA6/TR is a 6662 bp vector that expresses the Tet repressor under the control of the human CMV promoter. A figure summarizing the features of this vector can be downloaded by clicking here. The complete sequence for pcDNA6/TR is available for downloading from www.thermofisher.com or by contacting Technical Service.
TetR gene
The TetR gene used in pcDNA6/TR was originally isolated from the Tn10 transposon which confers resistance to tetracycline in E. coli and other enteric bacteria (Postle et al., 1984). The TetR gene from Tn10 encodes a class B Tet repressor and is often referred to as TetR(B) in the literature (Hillen and Berens, 1994).
The TetR gene encodes a repressor protein of 207 amino acids with a calculated molecular weight of 23 kDa. For more information about the Tet repressor and its interaction with the Tet operator, please refer to the review by Hillen and Berens (1994).
Features of pcDNA6/TR
The table below describes the relevant features of pcDNA6/TR. The vector includes the rabbit β-globin intron II to enhance expression of the TetR gene. For a more detailed description of the TetR gene and the Tet repressor, please refer to the previous page. All features have been functionally tested.
|Human cytomegalovirus (CMV) immediate early promoter
Permits high-level expression of the TetR gene (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987)
|Rabbit β-globin intron II (IVS)
|Enhances expression of the TetR gene (van Ooyen et al., 1979) in cultured cells
|TetR gene
|Encodes the Tet repressor that binds to tet operator sequences to repress transcription of the gene of interest in the absence of tetracycline (Postle et al., 1984; Yao et al., 1998)
|SV40 early polyadenylation signal
|Permits efficient transcription termination and polyadenylation of mRNA
|f1 origin
|Allows rescue of single-stranded DNA
|SV40 early promoter and origin
|Allows efficient, high-level expression of the blasticidin resistance gene in mammalian cells and episomal replication in cells expressing SV40 large T antigen
|EM7 promoter
|Synthetic prokaryotic promoter for expression of the blasticidin resistance gene in E. coli
|Blasticidin (bsd) resistance gene
|Allows selection of stable transfectants in mammalian cells (Kimura et al., 1994)
|SV40 early polyadenylation signal
|Allows efficient transcription termination and polyadenylation of mRNA
|pUC origin
|Permits high-copy number replication and growth in E. coli
|bla promoter
|Allows expression of the ampicillin (bla) resistance gene
Ampicillin ( bla) resistance gene
Allows selection of transformants in E. coli
Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert blasticidin S to a non-toxic deaminohydroxy derivative (Izumi et al., 1991).
Molecular weight, formula, and structure
The formula for blasticidin S is C17H26N8O5-HCl, and the molecular weight is 458.9. The diagram below shows the structure of blasticidin.
Handling Blasticidin
Always wear gloves, mask, goggles, and protective clothing (e.g. a laboratory coat) when handling blasticidin. Weigh out blasticidin and prepare solutions in a hood.
Preparing and storing stock solutions
Blasticidin is supplied in the T-REx Complete Kit, but may also be obtained separately from Invitrogen (Catalog no. R210-01) in 50 mg aliquots. Blasticidin is soluble in water. Sterile water is generally used to prepare stock solutions of 5 to 10 mg/ml.
- Dissolve blasticidin in sterile water and filter-sterilize the solution.
- Aliquot in small volumes suitable for one time use (see next to last point below) and freeze at -20°C for long-term storage or store at +4°C for short-term storage.
- Aqueous stock solutions are stable for 1-2 weeks at +4°C and 6-8 weeks at -20°C.
- pH of the aqueous solution should be 7.5 to prevent inactivation of blasticidin.
- Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer).
- Upon thawing, use what you need and store the thawed stock solution at +4°C for up to 2 weeks.
- Containing blasticidin may be stored at +4°C for up to 2 weeks.
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- Kozak, M. (1990). Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305.
- Laughon, A., and Gesteland, R. F. (1984). Primary Structure of the Saccharomyces cerevisiae GAL4 Gene. Mol. Cell. Biol. 4, 260-267.
- Lillie, J. W., and Green, M. R. (1989). Transcription Activation by the Adenovirus E1a Protein. Nature 338, 39-44.
- Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R., and Pluckthun, A. (1997). Specific Detection of His-tagged Proteins With Recombinant Anti-His Tag scFv-Phosphatase or scFv-Phage Fusions. BioTechniques 22, 140-149.
- Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S. C. (1992). DNA Recognition by GAL4: Structure of a Protein-DNA Complex. Nature 356, 408-414.
- McKnight, S. L. (1980). The Nucleotide Sequence and Transcript Map of the Herpes Simplex Virus Thymidine Kinase Gene. Nucleic Acids Res. 8, 5949-5964.
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- Misrahi, M., Atger, M., d'Auriol, L., Loosfelt, H., Meriel, C., Fridlansky, F., Guiochon-Mantel, A., Galibert, F., and Milgrom, E. (1987). Complete Amino Acid Sequence of the Human Progesterone Receptor Deduced from Cloned cDNA. Biochem. Biophys. Res. Commun. 143, 740-748.
- Mulsant, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1988). Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells. Somat. Cell Mol. Genet. 14, 243-252.
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Seattle Public Schools will open Lincoln High School in September 2019. Lincoln will be an attendance area high school serving students based on where they live. This requires drawing an attendance area for the new high school and reassigning students from other high school areas.
Ingraham High School will also have a significant capacity increase in 2019 with the construction of an addition. This is also being considered as new high school boundaries are drawn.
Thank you to the many families who attended regional open house meetings to talk about the boundary change process. We are continuing to seek feedback as we make additional adjustments to the boundaries before bringing a proposal to the School Board for approval.
This survey is an opportunity for you to share your top priorities as we make adjustments to the current boundary scenarios and provide any additional feedback that should be considered by district staff and the School Board. The listed priorities include those developed by the High School Boundary Task Force and values shared by families, staff and students at the recent community meetings.
The survey deadline has been extended. The High School Boundaries Community Survey will close Fri., Dec. 1, 2017. The online survey is available in English, Chinese, Spanish, Somali and Vietnamese. Participants who would benefit from using translated text can use the drop down menu in the upper right to enable the translated survey.
Take the High School Boundaries Community Survey
Adjustments will be made to the boundary scenarios based on feedback from families via email, the survey, open house meetings, and other channels. Adjustments will also be made in response to any School Board approved changes to the Highly Capable (HC) pathways. If changes are made to high school HC pathways they will be implemented in the 2019-20 school year in alignment with the opening of Lincoln High School.
It is anticipated that a proposal will be presented at the School Board Operations Committee meeting on December 7, 2017 and final approval of the boundary changes will take place on January 17, 2018.
For more background information on the high school boundary change process visit our:
For questions or comments please email firstname.lastname@example.com.


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Kids Walking Shoes
Here we offer comfortable, stylish and high-quality leisure footwear for kids, made by the world's famous sportswear brands like Nike, adidas, Reebok, Puma, New Balance, etc. The sports shoes will provide a maximum comfort, cushioning, stability and protection, while the high-quality materials will ensure great durability. A lightweight mesh upper will increase breathability and comfort by efficiently evaporating the excess moisture. Some of the models will have advanced midsole technology for additional amortization and responsiveness. We guarantee a wide product assortment, low prices and good service, quick package handling and delivery.
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Bobbie Burrell wasn't always a jazz singer, nor was she also an actress and a playwright.
Before she retired six years ago to Sarasota, Burrell, 66, worked as an accountant for nonprofit organizations in Washington, D.C., where she lived with her husband and raised two sons.
Sure, she grew up singing alongside her mother in a synagogue chorus and, later, in hootenanny folk jams as an undergrad at the University of Miami, but she was always too terrified to stand alone behind the microphone.
Friendly and easygoing, Burrell spent her 20s hanging out with singer/songwriters who had fled New York City for Miami's laid-back folk-music scene, singers such as David Crosby, Vince Martin and Fred Neil.
Even as she traveled in these circles, wistfully strumming her guitar with friends and listening to jazz records in her apartment, Burrell never had the confidence to take it further. Like many of us, she remained in her comfort zone, secretly wishing she was on stage.
Then, in 1996, she was diagnosed with breast cancer — and everything changed.
"It was like this catalyzing event," Burrell says. "I said, 'Well, now wait a minute. If this is what's going to happen, then I'm going to do all the things I always dreamed about.'"
After undergoing six months of chemotherapy and 11 hours of surgery, Burrell signed up for classes at the Elliston Studio for Jazz Studies, in Washington, D.C., where she studied a variety of styles and grew comfortable with scat, improvisation and vocal licks.
By her eighth week, she was singing onstage in D.C.'s Twins Lounge accompanied by legendary jazz pianist Ron Elliston. The performance was the final step in the Elliston program and a sort of reawakening for Burrell.
She began singing at other clubs around Washington, D.C., and at the East Coast Jazz Festival, in Maryland.
"In my experience, I've always needed to get sick or do something big before I could quit a job or move on," Burrell says. "Cancer was the biggest 2-by-4, smack-over-the-head that got me to move on."
When she was going through chemo, a friend encouraged her to see a Chinese Buddhist master who specialized in Eastern medicine. Though there was no guarantee that the master — a highly sought-after alternative healer in California — would meet with her, Burrell went for it, anyway.
To her delight, the master asked to see her for two sessions. So, Burrell purchased a plane ticket and rolled with it. At the end of the two days, when she asked the Buddhist master why he agreed to meet with her, he told her they knew each other 10,000 years ago.
"The experience," says Burrell, "was transformational."
But, 10 years later, the cancer came back.
By then, Burrell was living in Sarasota, where she underwent another six months of chemo. Rather than dwell on the reoccurrence, the singer contacted local jazz pianist Mike Markaverich, who played regular gigs at Caragiulos. The two went into the studio to record 14 jazz tracks for Burrell's first album, "Undecided."
She was bald and struggling to maintain her breath control, but the distraction, says Burrell, was "the best medicine ever."
Around this same time, Jeffery Kin, The Players Theatre's artistic director, had issued a call to local playwrights for his first Got A Minute 60-Second Play Festival. The proceeds from the show would be donated to the Susan G. Komen Breast Cancer Foundation. The e-mail was forwarded to Burrell.
"I'd never written a play before, but I figured I could do a minute," Burrell says. "So I wrote two. One was related to my breast cancer and the other was about life. I had no idea they would accept them."
Now in its fifth season, the popular summer play festival has raised more than $75,000 for cancer research. Burrell, who has been involved in every show since, is also on this year's bill. She'll sing a Ray Charles tune before intermission.
The festival opened other doors for the singer.
In 2008, she played the overbearing Mae Peterson in The Players' production of "Bye Bye Birdie," followed by a tough-talking restaurant owner in Kin's "The Spitfire Grill."
"Bobbie is such a beautifully balanced person," Kin says. "Her dark times have given her great perspective. She's able to step back and not let the minutia make her crazy."
Burrell does indeed sparkle with inner peace, the root of which could be any number of things, starting with the fact that after spending years as an accountant in a singer's body — an existence she jokingly calls "schizophrenic" — she's finally followed her passion.
"I told Jeffery once that inside I was a wuss, but that I was an empress, too," Burrell says. "And he looked at me and he said, 'That makes you an 'empuss,' which pretty much said it all. I don't look back and wish I had done this sooner, because when I sing now, I sing from a place of absolute freedom. It's a spiritual thing. My voice comes from a place of wholeness."
The Got A Minute 60-Second Play Festival is at 8 p.m. Friday Aug. 27, 8 p.m. Saturday, Aug. 28 and 2 p.m. Sunday Aug. 29, at The Players Theatre. Tickets are $20 to $30. All proceeds benefit Moffitt Cancer Center Foundation. For more information, call 365-2494 or visit theeclectictheatrecompany.com.


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One of the goals of this site is to promote critical discussion and debate on US foreign policy and the Israeli-Palestinian conflict. We want Mondoweiss to be a place that everyone feels comfortable visiting, to read and comment, regardless of political perspective. People might not always like what we post, but everyone should feel invited and encouraged to join the discussion, share their opinions, and engage in debate.
For this reason here are the ground rules:
1. No racist or sexist comments. This includes anti-Arab, Islamophobic and anti-Semitic comments (and yes, Christian-bashing too). This includes comments that disparage, intimidate or attack a person based on perceived ethnicity or gender.
2. No Nakba or Holocaust denial. We're not going to tolerate any discussion of the Jewish role in the rise of the Nazis which is used as a pretext for blaming Jews for the Nazi rise, a form of Holocaust denial we want no part of. Similarly, this policy includes Nakba denial as well, and efforts to blame the expulsion of Palestinians in 1948 on Palestinian actions.
3. No profanity. Along with racism and sexism the prevalence of profanity in the comments on the blog has forced people away. This has to end.
4. No personal attacks. We encourage spirited, passionate debate, but if you have to resort to vicious personal attack, you're not advancing the discussion. Stay on the issues.
5. No calls for violence or glorification or celebration of violence. Against anyone. Ever.
6. No imposture. You can use any pseudonym you like, but if you represent yourself as someone you're not, you're outta here.
7. No trolling. Wikipedia defines trolling as "someone who posts inflammatory, extraneous, or off-topic messages in an online community, such as an online discussion forum, chat room, or blog, with the primary intent of provoking other users into a desired emotional response." That definition is good enough for us. We hope our comment section can feature an engaged and free flowing debate, but we are not interested in commenters whose only aim is to disrupt or sabotage the discussion.
8. This is not a site to discuss 9/11 theories. This discussion turns into a huge distraction, is not central to the life of the site, and is a drag on the moderators.
If we judge that you have broken one of these rules you will be banned. We will ban the IP address you are posting from and if you try to post from another IP address we will ban that as well.
We like to think that this site has become a dynamic and necessary forum. Your help will ensure that it continues to grow and develop.
Phil & Adam


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IDE Controller
|Alfa Data: CD1200
|Connection: PCMCIA
CD1200, Front
CD1200, IDE connector
Hi Res Version, CD1200, Front - 184K
Hi Res Version, CD1200, IDE connector - 191K
A CD-ROM controller which connects to the PCMCIA slot of the A600 and A1200. It was a joint venture between BSC and Alfa Data. In the early days before the ATAPI standard was ratified, many manufacturers produced their own standards and protocols for adding CD-ROMs to computers. These custom controllers were generally found on old PC sound cards. The custom controllers looked like an IDE controller but were not compatible and were usually made by Sony, Panasonic or Mitsumi. The CD1200 is actually a Mitsumi controller and is designed for use with Mitsumi CD-ROMS that support the (now very obsolete) Mitsumi standard. Mitsumi also make standard IDE/ATAPI CDROMs. Another card for the Amiga which also supports the Mitsumi standard is the Tandem. It is possible to use standard APAPI CDROM's with this unit. Some people have reported that this works with the original drivers whilst others have said you need the ATAPI drivers for the IDE-Fix package.
Contributions to this page by:
Luis Alberto Francischini, Mate Sebok, Pawel Chwalowski, RiWa & Friends, Sebastian (Exin), Tomasz Baran
Letzte Änderung des Artikels: 2009-01-25 21:37
Revision: 1.0
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Prince William and Kate Middleton were huge fans of the Summer Games in London, but the royals are also coming out to show their support for the Paralympics during the opening ceremony.
No, Prince Harry was not present.
Supporters strip down for Prince Harry
U.S. & World
Wills donned a tailored navy suit with a turquoise tie to match those baby blue eyes of his, while Kate chose a shimmery, ivory and gold coat dress by Danish Label DAY Birger et Mikkelsen, a look she has worn twice before: once in 2010 for the wedding of Mel Nicholson and Oli Baker at St. Andrews, and then again for the wedding of Zara Phillips in July of last year.
Recycling never looked so glam, right?
Joining them in their booth were British Prime Minister David Cameron and his wife Samantha, Princess Anne, Prince Edward, Mayor Boris Johnson and, of course, Queen Elizabeth, sans hoodie.
We have a guess of who she might have loaned it to.


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Everyone is getting in on the newfangled mascara craze, aren't they? There are so many new mascaras being released lately that even a true fanatic like me can't keep up.
I'm one of those people who think that your mascara makes or breaks your look. I'm also one of those people who slather on a pint of inky black mascara before ever thinking of leaving the house - the more, the better, as long as it's good stuff and well applied.
What I love about Lash Stiletto: The brush isn't plastic! I'm tired of plastic or silicone brushes. I want the old fashioned spooley brushes that gave me tons of product on each swipe. I don't mind combing out the clumps, if there are any. The packaging is super cute. The smell is pleasant - some sort of fragrance is in it, but I can't put my finger on exactly what it is. Amazing lengthening properties. If you're looking for length, this is the mascara for you. The price is absolutely right - you'll only pay about $7.50 at a local drugstore for this one.
What I don't love about Lash Stiletto: It doesn't do a thing for volume. Amazing lengths, but not so much in the volume department. However, this isn't billed as a volumizing mascara, but don't make the mistake of thinking that you'll get some volume out of it. You won't. You will, however, get amazing long, skinny lashes.
Read more about chosing the right mascara for you with my "Of Course I Can Help You Choose a Mascara" article.
Read more Lash Stiletto reviews at Total Beauty.
Product Review: Maybelline New York Lash Stiletto
Everyone is getting in on the newfangled mascara craze, aren't they? There are so many new mascaras being released lately that even a true fanatic like me can't keep up.
Are Five Blades on a Razor Overkill?
I've been using the Gillette Venus Breeze for a long time, and figured the last thing I needed was more blades on my razor. I suffer from ingrown hairs occasionally (I know, who doesn't? But if you have curly hair like me, you know exactly what I'm talking about), and the advice has always been to use a single blade razor. Well, I'm not down with that - I have a limited amount of time that I'm willing to invest in shaving, so my triple blades have suited me just fine.
Recently, Gillette released their newest addition to their line, the Venus Embrace Razor, which has a whopping five blades to it. Contrary to what I expected, this razor did an excellent job giving me a smooth shave without any resulting problems such as razor burn or ingrown hairs.
You can pick one up at any major retailer for about $10.


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Our mission presents a profound intellectual challenge due to the complexity, multiplicity, and interconnectedness of the climate system itself. What is the present state of our climate, and what causes it to change? Does the system reverberate chaotically and recombine into a new, different state? Or do external forcings such as alterations in the incidence of solar radiation and increased carbon dioxide levels prevail as agents for change? What is the combined effect of forces both internal and external acting simultaneously? And then there is the problem of timescales. We know from proxy measurements that the climate has lurched many times between glacial and interglacial states, but how do we separate long-term trends from short-term variations? Then, contributing to the complexity and blurring the difference between causality and coincidence, there are numerous, quasi-predictable cycles in the atmosphere and ocean, such as El Nino and the North Atlantic Oscillation, capable of changing weather conditions worldwide.
Industrialization and its demand for natural resources, continental-scale agriculture and its demand for land, and the swelling global population with its demand for everything continue to reshape the Earth. Since the dawn of the Industrial Revolution, our exhausts and effluviums have increased atmospheric carbon-dioxide levels by more than one third. These products of human activity have begun to leave their mark, and are changing our climate. What separates anthropogenic climate change from natural climate variations is its unprecedented rate of change. Not only by changing Earth's climate but by doing so at fast-forward speed, humankind has become a true geophysical force.
It is of course insufficient for a scientist simply to say that humanity – or humanity in conjunction with other forces—has changed the world's climate. As scientists, we need to identify the causes of change and, if possible, need to predict change. But to do so requires a degree of understanding of the climate system as a whole far greater than we possess at present. And that brings us back to our mission and the pressing intellectual challenge before us. To fulfill our mission by confronting the challenge, we will approach the problem in the light of two guiding questions:
How susceptible is the Earth system to perturbations?
What are the limits of Earth system predictability?
We are keenly aware of the challenges that must be overcome to enhance our understanding of Earth's dynamic climate system. But the challenges are not daunting – intellectual and technical opportunities abound as the new decade unfolds.
Our greatest opportunity lies in the advancement of comprehensive Earth system modeling. We will build on the history of research within the MPI-M that started from the development of component models of the atmosphere and ocean, moved to pioneering studies of the coupled atmosphere-ocean system, and recently culminated in the development of the MPI Earth system model. However, increasing further the complexity of our models will not by itself provide answers to our guiding questions; instead, answers will arise from the richness and clarity of the concepts that our models help us create.
The MPI-M continues to attract the most talented and creative scientists from around the world. We integrate them into a stimulating institutional environment, which consists of excellent support staff, state-of-the-art facilities, and the MPI-M membership in the Cluster of Excellence CliSAP.
The MPI-M requires and enjoys privileged access to high-performance computing facilities, chief among them the German Climate Computing Centre (DKRZ). The ever-increasing computational capacity makes some tasks easier and others imaginable. For example, the exploration of climate predictability requires the simulation of large ensembles of runs, each differing by a small amount in the initial conditions; this task is greatly aided by a sufficiently powerful computing and data storage system such as at DKRZ. Other simulations have only now become possible, such as replacing cumulus parameterizations by convection-resolving models and thus basing the simulation on much firmer conceptual grounds.
To evaluate and initialize our models, we will increasingly exploit the growing observational record, which allows us to extract fundamentally new insights into the changing Earth system; from the vertical structure of clouds, to the growing uptake of heat by the ocean, to ever richer descriptions of the land biosphere. The global observational record is complemented by a new generation of targeted measurement campaigns aiming at, for example, cloud dynamics in the subtropics or the ocean's meridional overturning circulation.
Opportunities abound. And we have in the Max Planck Institute for Meteorology a practiced, fully functioning institution with a thoroughgoing history in climate research. We have the required quality in our scientific and support staff to develop and use comprehensive Earth system models. We have the technical and scientific capability to augment and integrate into our models a rich and growing observational record. We understand our mission and the questions that must be addressed to realize it. And so, prepared and dedicated, we look forward to the coming decade.
From Strategic Plan "2020 Vision", page 5ff, 2011


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MLB: Ex-Dodgers owner may testify in fan beating case
LOS ANGELES — A lawyer for a San Francisco Giants fan who was attacked and gravely injured at Dodger Stadium plans to call former Dodgers owner Frank McCourt as a witness in a negligence lawsuit against the team and McCourt.
LOS ANGELES — A lawyer for a San Francisco Giants fan who was attacked and gravely injured at Dodger Stadium plans to call former Dodgers owner Frank McCourt as a witness in a negligence lawsuit against the team and McCourt.
However, McCourt's stay on the witness stand could be brief Friday if a judge limits issues allowed in the questioning.
Attorney Tom Girardi, who represents beating victim Bryan Stow, wants to question McCourt about team finances and to suggest that McCourt skimped on spending for security at the stadium while lavishing millions of dollars on his own lifestyle.
Stow, who became a symbol of violence at sporting events, suffered severe brain damage after being beaten by two Dodgers fans who pleaded guilty to criminal charges in the attack.
Testimony thus far at the civil trial has focused on Stow's injuries and need for lifetime care, and the contention that there was insufficient security to protect fans at the 2011 opening day game between the California rivals.
Witnesses have said no security guards were visible in the parking lot where Stow was beaten. The defense countered that there was more security than at any other Dodgers opening day in history.
Superior Court Judge Victor Chavez will decide at a special hearing whether McCourt must testify about team finances.
McCourt paid $430 million in 2004 to buy the team, Dodger Stadium and 250 acres of land that includes parking lots, from the Fox division of Rupert Murdoch's News Corp., a sale that left the team with about $50 million in cash at the time.
The Dodgers went into bankruptcy protection in June 2011 and the next year McCourt sold the team for $2 billion to a group that included former Los Angeles Lakers star Magic Johnson.
The group vowed to restore dignity to the storied franchise after the era of McCourt, who was widely reviled by Dodgers fans for driving the Dodgers to the brink of bankruptcy.
In bankruptcy filings, attorneys for Major League Baseball said McCourt looted more than $180 million in revenue from the club.