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Are you trying to sell stuff online?
I'm sure you will agree that converting your visitors into sales is hard.
And getting yourself profitable? That's even harder.
But there is a simple secret to increasing the value of every customer you sell to:
Sales funnels. Yes.Clickfunnels
By creating a well-designed sales funnel, it is possible to dramatically increase your earnings and provide more value for your customers.
In this article, I'm going to present you to ClickFunnels, the software I'm using to build sales funnels for my business.
You do not need any technical know-how to utilize this, and it is going to really change how you operate your business.
If you're prepared to throw yourself into the ClickFunnels world, it has one of the best affiliate programs out there. I will be talking about that too later on.
Here is what I will be covering in this ClickFunnels review.
- What is ClickFunnels?
- What are the best parts of ClickFunnels?
- What I don't like about ClickFunnels
- ClickFunnels Pricing
- Everything else you need to know about ClickFunnels
- How can you make ClickFunnels work for you?
- ClickFunnels versus the competition
- Pros and Cons of ClickFunnels
- Final Thoughts
What is ClickFunnels?
ClickFunnels was created back in 2014 by Russell Brunson and his team. Since that time, Russell has turned ClickFunnels to a $100 million a year company and they just crossed over 65,000 active members as of August 2018.
In its core, ClickFunnels is a software for constructing sales funnels. With a lot of additional bells and whistles.
You can use it to build:
– Landing pages
– Squeeze pages
– Webinars
– Membership sites
– Every kind of sales funnel you can think of, complete with order bumps, upsells and downsells
Additionally, with the highest end Etison Suite package you will even have the ability to start your own affiliate program (Backpack) and replace your email service with (Actionetics MD).
Basically, you're getting all you need to advertise and sell your products online, without needing to hire expensive developers.
And individuals are utilizing ClickFunnels with great success.
If you check out their 2 Comma Club website, you can see that almost 300 entrepreneurs have already created 1 million dollar funnels utilizing ClickFunnels.
That is amazing.Yes.Clickfunnels
What are the best parts of ClickFunnels?
Best Feature #1: Funnel template blueprints
If you have never created a sales funnel before then ClickFunnels has you covered.
Russell has identified 22 distinct types of funnels that he has used successfully in his own business.
The funnel forms fall into 3 main classes: Lead Capture Funnels, Sales Page Funnels, and Event Funnels. But there are a few additional special funnels like Membership funnels you can create too.
They include simple funnels such as the Squeeze Page Funnel which collects a visitor's email address and then sends them to a Thank you page when registering.
Or to more sophisticated sales funnels such as the 2-Step TripWire Funnel which sells a very low ticket front-end product (your tripwire) and then upsells them to your more expensive products using Order Form bumps, and One Click upsells or Downsells.
Moreover, every funnel type has its own set of free templates and paid templates. So even in the event that you don't have any design skills, your funnel pages will all have a consistent, professional design.
Here's a high-level overview of how to create your first sales funnel:
- Choose a funnel type.
- Select a template.
- Customize each page to fit your business.
- Add your products and ensure all your email and payment integrations are set up.
- Set a domain name for your new funnel.
- Save your funnel.
Your sales funnel is now live and you can start sending traffic to it. Yes.Clickfunnels
Best Feature #2: Good Visual Drag and Drop Editor
While ClickFunnels gives you a great starting point with its funnel and page templates, the ClickFunnels editor is where you can truly make each page in your funnel your own.
Pages in ClickFunnels are laid out with sections that can have any number of rows and columns. You can then use your mouse to drag elements around and place them where you need them.
While not as flexible as Instapage's landing page editor, it is still quite good and it may be used to achieve pretty much any design you desire.
What I like is that the editor is quite intuitive to use.
Selecting elements on the page and moving them where you want is very seamless.
Text elements can be edited inline, and modifying any element's properties can be accomplished via the fly-out sidebar.
And unlike other editors out there, everything is organized in a logical manner. You are not spending a lot of time hunting for what you would like to do.
The main thing is that no coding is required. So you don't need to spend time studying HTML or CSS or any technical things like that.
Best Feature #3: Nice Selection of Page Elements
Elements (or widgets) are vital for providing you the flexibility to design the exact pages you woud like and ClickFunnels provides you a large array of them to use.
Basic elements comprise of Headline, Image, Text, Button, Input types and Video widgets. These are the fundamental building blocks for any landing page you construct in the software.
ClickFunnels also includes a number of advanced components like:
– SMS Signup
– Surveys
– Pricing Tables
– Progress Bars
– Facebook Comments
– FAQ Blocks
– Countdown Timers
– Custom HTML
Additionally, there are specialized elements like Membership elements for your membership pages and order and shipping form elements for your order pages.
Each element can be easily customized from the editor sidebar. With majority of elements, you can change common properties such as background colors, margins, fonts, and orientation.
Then there are element specific properties like the image URL for the Image element or the Button text of your button.
Best Feature #4: The ClickFunnels Affiliate program is awesome
There is little doubt in my mind that ClickFunnels has the best, legitimate affiliate marketing program that you can be a part of.
The Fundamentals:
The ClickFunnels affiliate program pays 40% recurring commissions for each referral you make and you can win cool prizes like $500 towards your own dream car as soon as you hit 100 active referrals.
In addition to that, they offer 5%, 2-tier commissions for referring people to the ClickFunnels affiliate program itself.
In addition you earn 40% commissions by promoting any product in the ClickFunnel's ecosystem such as DotCom Secrets, Expert Secrets or even the Software Secrets books.
And thanks to the ClickFunnel's sticky cookie, you will also earn commissions on any upsells your referrals buy as they go through their specific funnels too.
That is not all:
ClickFunnels also has another excellent feature for affiliates known as share funnels.
Within the Settings tab of any of your funnels, you can grab your Share Funnel URL.
When someone visits this URL, they'll be able to bring this funnel directly into their accounts if they are an existing ClickFunnels member. If they aren't a member yet, they can sign up for the ClickFunnels 14-day free trial and then get your funnel added to their accounts.
What's really awesome is that this share URL also functions as your affiliate link, and you will get a recurring commission if this new user ends up staying.
What I don't like about ClickFunnels?
Bad Part #1: Why is adding a funnel so slow?
After picking your funnel type and template, it needs to get added to your account.
For some reason, this procedure appears to take approximately 8-10 seconds each time.
While I don't know what is really going on behind the scenes, it does not make sense to me why this could take so long. It's somewhat annoying, although only a minor inconvenience.
Bad Part #2: Limits on funnels you can create and visitors
ClickFunnels limits you to 20 funnels, 100 pages, and 20,000 visitors in their cheapest plan. I am really not a fan of software products which limit you in this way.
If you're just building sales funnels for yourself, these limits are probably okay.
However you can get more annoyed at such limitations if you're doing client work, creating funnels for multiple customers or promoting ClickFunnels as an affiliate with share funnels.
You will need to upgrade to the Etison Suite plan if you would like to build unlimited funnels.
Bad Part #3: Fairly High learning curve
The scope of what ClickFunnels can do is fairely huge, and it can certainly be intimidating to some.
That's why ClickFunnels put so much work into the onboarding process to help ease you into the software. The ClickFunnels interface is pretty well made for the most part and once you've assembled a couple funnels yourself, the whole procedure does get easier over time.
ClickFunnels Pricing
Over time, ClickFunnels has evolved their pricing plans. In the beginning, they had 3 different pricing tiers, but they have now been consolidated into 2 separate plans.
So how much is ClickFunnels now?
At the moment, the cheapest ClickFunnels plan is $97 a month. On this plan, you get access to all the funnel creation tools, but you are limited to generating 20 funnels, 100 pages, and 20,000 visitors. You also don't get access to Actionetics, their built-in email service provider and Backpack, which lets you create your own affiliate program.
To gain access to that, you'll need to go for the Etison Suite plan. It features everything, plus you receive unlimited funnels, pages, and visitors. However, this will run you $297 a month.
As far as I can tell, there aren't any ClickFunnels coupon codes available. However, you can buy Funnel Hacks for $997 which gives you 6 months of Etison Suite for free plus some extra bonuses. This brings the cost down to $166.16 per month.
ClickFunnels does make it easy for you to demo their service with their free, 14-day free trial. It's not limited whatsoever which lets you give the software a complete test before you fully commit.
If $97 or $297 a month seems like too big a commitment for your current budget, there's also a semi-secret, unadvertised Share Funnel plan that you can also have access to for only $19 a month. You can only create 3 funnels and 10 pages with this plan, but it's a great way to try out ClickFunnels on a budget.
Everything else you need to know about ClickFunnels
How is their support?
ClickFunnels support is fairly good in my experience, even though they can be a little slow in responding. They utilize Intercom, which means you can directly ask questions in the live chat box. Support requests are usually answered within a few hours and I found the staff to pretty helpful to all my queries.
Do I need my own domain name? Yes.Clickfunnels
You don't necessarily need your own domain name to launch a funnel, although I would advise it. If you do not have one then your funnel URL will look something like http://username.clickfunnels.com.
One nice bonus that I did not know about is that ClickFunnels will get you your first domain name for free and even set up SSL on it for you. You can discover how to do this from the first game in the onboarding training.
Do they have an active community?
ClickFunnels has an massive community. There is the official group on FB with over 100k members and the ClickFunnels Avengers FB group (for affiliates) that has over 30k members.
Both groups are very active and it is a great place to get opinions on your sales funnels and answer any questions that you might have.
Additionally, there are several other FB groups run by ClickFunnels affiliates that are also quite active. The main one I suggest joining is Automation Nation. It's run by Spencer Mecham who is ClickFunnel's top affiliate.
What integrations do they offer?
Even though ClickFunnels has its own email service provider via Actionetics, it also integrates directly with all the best email marketing software out there.
For payment gateways, ClickFunnels integrates with Stripe, Paypal, Clickbank, Infusionsoft, Authorize.net, and a few others. The one limitation with using Paypal though is that it does not support One Click Upsells or One Time Offers. That's why I would highly recommend going with Stripe if you're able to.
What is the Funnel Hacks system?
If you are looking to get the $297/mo. Etison Suite plan, then it's definitely worth it to join with Funnel Hacks instead to save some additional money. For $997, you are going to get six months of the Etison Suite plan which is $1,782 in value.
You will also get a bunch of additional bonuses and training that include:
– 6 Week Funnel Hacks Master Class ($2,997 value)
– Instant Traffic Hacks ($1,997 value)
– Inception Secrets ($1,997 value)
– SOAP & Seinfeld Email Sequences ($997 value)
Is ClickFunnels a scam?
I believe ClickFunnels gets some negative buzz around it because of all the affiliates who are making an effort to market it.
However, no, ClickFunnels is not a scam. It's certainly the real deal.
It's a great software with an amazing affiliate opportunity. So many people are using ClickFunnels to build 6 and 7 figure businesses.
It's a software with an amazing affiliate opportunity. So many men and women are utilizing ClickFunnels to build 6 and 7 figure companies.
When people ask, "How can I create a sales funnel", ClickFunnels is the #1 response hands down. There's no other application that can do every thing that ClickFunnels does at the moment.
What else is ClickFunnels used for?
Besides creating standard sales funnels, there are many ways marketers are using ClickFunnels right now in their business.
It is terrific for creating opt-in funnels to help you generate more prospects and build your email list.
You can produce webinar funnels to help you develop your brand and sell your products and services.
And you can use it to build membership sites where you can gate your premium content so that it is accessible only to subscribers.
How can you make ClickFunnels work for you? Yes.Clickfunnels
To start with, you need to have a product or service to market. If you don't have something to market, then don't even bother looking into ClickFunnels until you do. Unless you just want to market it as an affiliate.
Next, you need to be willing to commit time and money into creating your value ladder and constructing your entire sales funnel.
That means creating a lead magnet or low-cost tripwire product to get customers or clients into your funnel. And coming up with appropriate upsells, order bumps and other OTOs that complement or improve your principal product offering.
If you are unclear how to start, I would take a look at Russell's very own multi-million dollar sales funnels to provide you with inspiration. Read his book DotComSecrets where he explains exactly the why and how of creating sales funnels and how to tell stories to sell your products and services.
And as you go through his own sales funnels yourself you can see the way he goes from low-cost tripwire (DotComSecrets) to promoting his flagship product (ClickFunnels).
By building his very own community of raving fans he can then upsell his various different products and events like Funnel Scripts, Funnel Hackers Live, and more.
As soon as you've gone through his funnels, then you can take the parts that apply to your own company and create a strategy for your own sales funnel. Once you have that in place, you'll be prepared to begin making money with ClickFunnels.
ClickFunnels versus the competition
There are a couple of ClickFunnels alternatives out there which share a number of the same capabilities as ClickFunnels. Two that I use myself are LeadPages and SamCart.
ClickFunnels vs LeadPages
Today, LeadPages is one of the market leaders in the landing page builder space and it's a very popular tool in its own right.
It also recently added the capability to sell products directly on their pages using its new Checkout widget
Each product comes with a large selection of fine looking templates. Both have drag and drop editors which make it effortless for you to build the designs you desire. And both integrate with the popular email service providers.
But, Leadpages is more limited in scope compared to ClickFunnels. You use it to create landing pages that will allow you to collect leads. This is not a bad thing because LeadPages really shines for building landing pages fast.
At this time, one of the best strategies for developing your email list is by using content upgrades. By creating targeted lead magnets into your most well-known articles you can get opt-in conversion rates that far surpass your typical pop-up.
LeadPages is the best service for implementing this strategy with its LeadBoxes, 2 step opt-ins, and automatic lead magnet delivery which is why they are the most popular popup you will see now.
So if you're primarily concentrated on lead generation, I would definitely suggest trying out LeadPages.
However, if you are selling a product or service, then ClickFunnels is the better choice. While it comes with a higher learning curve than Leadpages, once you master the tool, you can really explode your sales once you start creating sales funnels for each of your products.
However, if you are selling a service or product, then ClickFunnels is the better alternative . While it comes with a higher learning curve than Leadpages, as soon as you master the instrument, you can really explode your sales as soon as you begin creating sales funnels for each of your products.
ClickFunnels vs SamCart
SamCart is a product which allows users to build beautiful, high converting checkout pages. Which gives it a lot in common with ClickFunnels.
SamCart is targeted towards getting set up and selling fast. It supports both Stripe and Paypal and it includes 17 distinct checkout page templates which you can customize.
Concerning flexibility, there's absolutely no comparison between ClickFunnel's drag and drop editor and SamCart's.
ClickFunnels is infinitely more powerful in that you can design any checkout page you can dream of. SamCart only allows for some basic customizations to its own pre-made templates.
SamCart does permit you to add order bumps, upsells and downsells like ClickFunnels does. You can also quickly create coupons for your products which is just another great way to raise conversions.
However, where Samcart is lacking, is the ability to capture leads and build complete sales funnels. The products come in at the same $97 a month price point, and I find ClickFunnels to be quite a bit more flexible and more powerful than Samcart.
ClickFunnels Pros
– Best software for building marketing and sales funnels
– Editor balances ease of use with flexibility
– Integrates with all major email and payment providers
– Beautiful looking templates for every type of funnel
– Great on-boarding procedure for learning the program
– Software continues to get better after every update
– Readily share any funnels you create and automatically earn affiliate commissions
– 2-tier affiliate program
– Free domain name with SSL set up
– 14-day free trial
ClickFunnels Cons
– Limits on the number of funnels, pages, and visitors with the standard plan
– Plently of things to learn, a steep learning curve
– Split testing is pretty basic
Final Thoughts Yes.Clickfunnels
Which software you go with depends on your requirements and the quantity of work you're willing to invest in your enterprise.
Although many of you are selling standalone products, are you willing to put in the effort necessary to make a high converting sales funnel?
If not, ClickFunnels might be overkill for you. It's possible to collect leads or market your front end product more cheaply with alternative services.
However, if raising the average lifetime value of your clients is important to you, then I really suggest giving ClickFunnels a try.
Not just to access the software but also since the coaching that you get will probably be both inspirational and enlightening.
Make the most of the ClickFunnels 14-day free trial, and see whether you can use it to grow your business now.


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At all times, PostgreSQL maintains a write ahead log (WAL) in the
pg_wal/ subdirectory of the cluster's data directory. The log records every change made to the database's data files. This log exists primarily for crash-safety purposes: if the system crashes, the database can be restored to consistency by "replaying" the log entries made since the last checkpoint. However, the existence of the log makes it possible to use a third strategy for backing up databases: we can combine a file-system-level backup with backup of the WAL files. If recovery is needed, we restore the file system backup and then replay from the backed-up WAL files to bring the system to a current state. This approach is more complex to administer than either of the previous approaches, but it has some significant benefits:
We do not need a perfectly consistent file system backup as the starting point. Any internal inconsistency in the backup will be corrected by log replay (this is not significantly different from what happens during crash recovery). So we do not need a file system snapshot capability, just tar or a similar archiving tool.
Since we can combine an indefinitely long sequence of WAL files for replay, continuous backup can be achieved simply by continuing to archive the WAL files. This is particularly valuable for large databases, where it might not be convenient to take a full backup frequently.
It is not necessary to replay the WAL entries all the way to the end. We could stop the replay at any point and have a consistent snapshot of the database as it was at that time. Thus, this technique supports point-in-time recovery: it is possible to restore the database to its state at any time since your base backup was taken.
If we continuously feed the series of WAL files to another machine that has been loaded with the same base backup file, we have a warm standby system: at any point we can bring up the second machine and it will have a nearly-current copy of the database.
pg_dump and pg_dumpall do not produce file-system-level backups and cannot be used as part of a continuous-archiving solution. Such dumps are logical and do not contain enough information to be used by WAL replay.
As with the plain file-system-backup technique, this method can only support restoration of an entire database cluster, not a subset. Also, it requires a lot of archival storage: the base backup might be bulky, and a busy system will generate many megabytes of WAL traffic that have to be archived. Still, it is the preferred backup technique in many situations where high reliability is needed.
To recover successfully using continuous archiving (also called "online backup" by many database vendors), you need a continuous sequence of archived WAL files that extends back at least as far as the start time of your backup. So to get started, you should set up and test your procedure for archiving WAL files before you take your first base backup. Accordingly, we first discuss the mechanics of archiving WAL files.
In an abstract sense, a running PostgreSQL system produces an indefinitely long sequence of WAL records. The system physically divides this sequence into WAL segment files, which are normally 16MB apiece (although the segment size can be altered during initdb). The segment files are given numeric names that reflect their position in the abstract WAL sequence. When not using WAL archiving, the system normally creates just a few segment files and then "recycles" them by renaming no-longer-needed segment files to higher segment numbers. It's assumed that segment files whose contents precede the last checkpoint are no longer of interest and can be recycled.
When archiving WAL data, we need to capture the contents of each segment file once it is filled, and save that data somewhere before the segment file is recycled for reuse. Depending on the application and the available hardware, there could be many different ways of "saving the data somewhere": we could copy the segment files to an NFS-mounted directory on another machine, write them onto a tape drive (ensuring that you have a way of identifying the original name of each file), or batch them together and burn them onto CDs, or something else entirely. To provide the database administrator with flexibility, PostgreSQL tries not to make any assumptions about how the archiving will be done. Instead, PostgreSQL lets the administrator specify a shell command to be executed to copy a completed segment file to wherever it needs to go. The command could be as simple as a
cp, or it could invoke a complex shell script — it's all up to you.
To enable WAL archiving, set the wal_level configuration parameter to
replica or higher, archive_mode to
on, and specify the shell command to use in the archive_command configuration parameter. In practice these settings will always be placed in the
postgresql.conf file. In
%p is replaced by the path name of the file to archive, while
%f is replaced by only the file name. (The path name is relative to the current working directory, i.e., the cluster's data directory.) Use
%% if you need to embed an actual
% character in the command. The simplest useful command is something like:
archive_command = 'test ! -f /mnt/server/archivedir/%f && cp %p /mnt/server/archivedir/%f' # Unix archive_command = 'copy "%p" "C:\\server\\archivedir\\%f"' # Windows
which will copy archivable WAL segments to the directory
/mnt/server/archivedir. (This is an example, not a recommendation, and might not work on all platforms.) After the
%p and
%f parameters have been replaced, the actual command executed might look like this:
test ! -f /mnt/server/archivedir/00000001000000A900000065 && cp pg_wal/00000001000000A900000065 /mnt/server/archivedir/00000001000000A900000065
A similar command will be generated for each new file to be archived.
The archive command will be executed under the ownership of the same user that the PostgreSQL server is running as. Since the series of WAL files being archived contains effectively everything in your database, you will want to be sure that the archived data is protected from prying eyes; for example, archive into a directory that does not have group or world read access.
It is important that the archive command return zero exit status if and only if it succeeds. Upon getting a zero result, PostgreSQL will assume that the file has been successfully archived, and will remove or recycle it. However, a nonzero status tells PostgreSQL that the file was not archived; it will try again periodically until it succeeds.
When the archive command is terminated by a signal (other than SIGTERM that is used as part of a server shutdown) or an error by the shell with an exit status greater than 125 (such as command not found), the archiver process aborts and gets restarted by the postmaster. In such cases, the failure is not reported in pg_stat_archiver.
The archive command should generally be designed to refuse to overwrite any pre-existing archive file. This is an important safety feature to preserve the integrity of your archive in case of administrator error (such as sending the output of two different servers to the same archive directory).
It is advisable to test your proposed archive command to ensure that it indeed does not overwrite an existing file, and that it returns nonzero status in this case. The example command above for Unix ensures this by including a separate
test step. On some Unix platforms,
cp has switches such as
-i that can be used to do the same thing less verbosely, but you should not rely on these without verifying that the right exit status is returned. (In particular, GNU
cp will return status zero when
-i is used and the target file already exists, which is not the desired behavior.)
While designing your archiving setup, consider what will happen if the archive command fails repeatedly because some aspect requires operator intervention or the archive runs out of space. For example, this could occur if you write to tape without an autochanger; when the tape fills, nothing further can be archived until the tape is swapped. You should ensure that any error condition or request to a human operator is reported appropriately so that the situation can be resolved reasonably quickly. The
pg_wal/ directory will continue to fill with WAL segment files until the situation is resolved. (If the file system containing
pg_wal/ fills up, PostgreSQL will do a PANIC shutdown. No committed transactions will be lost, but the database will remain offline until you free some space.)
The speed of the archiving command is unimportant as long as it can keep up with the average rate at which your server generates WAL data. Normal operation continues even if the archiving process falls a little behind. If archiving falls significantly behind, this will increase the amount of data that would be lost in the event of a disaster. It will also mean that the
pg_wal/ directory will contain large numbers of not-yet-archived segment files, which could eventually exceed available disk space. You are advised to monitor the archiving process to ensure that it is working as you intend.
In writing your archive command, you should assume that the file names to be archived can be up to 64 characters long and can contain any combination of ASCII letters, digits, and dots. It is not necessary to preserve the original relative path (
%p) but it is necessary to preserve the file name (
Note that although WAL archiving will allow you to restore any modifications made to the data in your PostgreSQL database, it will not restore changes made to configuration files (that is,
pg_hba.conf and
pg_ident.conf), since those are edited manually rather than through SQL operations. You might wish to keep the configuration files in a location that will be backed up by your regular file system backup procedures. See Section 20.2 for how to relocate the configuration files.
The archive command is only invoked on completed WAL segments. Hence, if your server generates only little WAL traffic (or has slack periods where it does so), there could be a long delay between the completion of a transaction and its safe recording in archive storage. To put a limit on how old unarchived data can be, you can set archive_timeout to force the server to switch to a new WAL segment file at least that often. Note that archived files that are archived early due to a forced switch are still the same length as completely full files. It is therefore unwise to set a very short
archive_timeout — it will bloat your archive storage.
archive_timeout settings of a minute or so are usually reasonable.
Also, you can force a segment switch manually with
pg_switch_wal if you want to ensure that a just-finished transaction is archived as soon as possible. Other utility functions related to WAL management are listed in Table 9.87.
wal_level is
minimal some SQL commands are optimized to avoid WAL logging, as described in Section 14.4.7. If archiving or streaming replication were turned on during execution of one of these statements, WAL would not contain enough information for archive recovery. (Crash recovery is unaffected.) For this reason,
wal_level can only be changed at server start. However,
archive_command can be changed with a configuration file reload. If you wish to temporarily stop archiving, one way to do it is to set
archive_command to the empty string (
''). This will cause WAL files to accumulate in
pg_wal/ until a working
archive_command is re-established.
The easiest way to perform a base backup is to use the pg_basebackup tool. It can create a base backup either as regular files or as a tar archive. If more flexibility than pg_basebackup can provide is required, you can also make a base backup using the low level API (see Section 26.3.3).
It is not necessary to be concerned about the amount of time it takes to make a base backup. However, if you normally run the server with
full_page_writes disabled, you might notice a drop in performance while the backup runs since
full_page_writes is effectively forced on during backup mode.
To make use of the backup, you will need to keep all the WAL segment files generated during and after the file system backup. To aid you in doing this, the base backup process creates a backup history file that is immediately stored into the WAL archive area. This file is named after the first WAL segment file that you need for the file system backup. For example, if the starting WAL file is
0000000100001234000055CD the backup history file will be named something like
0000000100001234000055CD.007C9330.backup. (The second part of the file name stands for an exact position within the WAL file, and can ordinarily be ignored.) Once you have safely archived the file system backup and the WAL segment files used during the backup (as specified in the backup history file), all archived WAL segments with names numerically less are no longer needed to recover the file system backup and can be deleted. However, you should consider keeping several backup sets to be absolutely certain that you can recover your data.
The backup history file is just a small text file. It contains the label string you gave to pg_basebackup, as well as the starting and ending times and WAL segments of the backup. If you used the label to identify the associated dump file, then the archived history file is enough to tell you which dump file to restore.
Since you have to keep around all the archived WAL files back to your last base backup, the interval between base backups should usually be chosen based on how much storage you want to expend on archived WAL files. You should also consider how long you are prepared to spend recovering, if recovery should be necessary — the system will have to replay all those WAL segments, and that could take awhile if it has been a long time since the last base backup.
The procedure for making a base backup using the low level APIs contains a few more steps than the pg_basebackup method, but is relatively simple. It is very important that these steps are executed in sequence, and that the success of a step is verified before proceeding to the next step.
Low level base backups can be made in a non-exclusive or an exclusive way. The non-exclusive method is recommended and the exclusive one is deprecated and will eventually be removed.
A non-exclusive low level backup is one that allows other concurrent backups to be running (both those started using the same backup API and those started using pg_basebackup).
Ensure that WAL archiving is enabled and working.
Connect to the server (it does not matter which database) as a user with rights to run pg_start_backup (superuser, or a user who has been granted EXECUTE on the function) and issue the command:
SELECT pg_start_backup('label', false, false);
label is any string you want to use to uniquely identify this backup operation. The connection calling
pg_start_backup must be maintained until the end of the backup, or the backup will be automatically aborted.
By default,
pg_start_backup can take a long time to finish. This is because it performs a checkpoint, and the I/O required for the checkpoint will be spread out over a significant period of time, by default half your inter-checkpoint interval (see the configuration parameter checkpoint_completion_target). This is usually what you want, because it minimizes the impact on query processing. If you want to start the backup as soon as possible, change the second parameter to
true, which will issue an immediate checkpoint using as much I/O as available.
The third parameter being
false tells
pg_start_backup to initiate a non-exclusive base backup.
Perform the backup, using any convenient file-system-backup tool such as tar or cpio (not pg_dump or pg_dumpall). It is neither necessary nor desirable to stop normal operation of the database while you do this. See Section 18.104.22.168 for things to consider during this backup.
In the same connection as before, issue the command:
SELECT * FROM pg_stop_backup(false, true);
This terminates backup mode. On a primary, it also performs an automatic switch to the next WAL segment. On a standby, it is not possible to automatically switch WAL segments, so you may wish to run
pg_switch_wal on the primary to perform a manual switch. The reason for the switch is to arrange for the last WAL segment file written during the backup interval to be ready to archive.
pg_stop_backup will return one row with three values. The second of these fields should be written to a file named
backup_label in the root directory of the backup. The third field should be written to a file named
tablespace_map unless the field is empty. These files are vital to the backup working and must be written byte for byte without modification, which may require opening the file in binary mode.
Once the WAL segment files active during the backup are archived, you are done. The file identified by
pg_stop_backup's first return value is the last segment that is required to form a complete set of backup files. On a primary, if
archive_mode is enabled and the
wait_for_archive parameter is
pg_stop_backup does not return until the last segment has been archived. On a standby,
archive_mode must be
always in order for
pg_stop_backup to wait. Archiving of these files happens automatically since you have already configured
archive_command. In most cases this happens quickly, but you are advised to monitor your archive system to ensure there are no delays. If the archive process has fallen behind because of failures of the archive command, it will keep retrying until the archive succeeds and the backup is complete. If you wish to place a time limit on the execution of
pg_stop_backup, set an appropriate
statement_timeout value, but make note that if
pg_stop_backup terminates because of this your backup may not be valid.
If the backup process monitors and ensures that all WAL segment files required for the backup are successfully archived then the
wait_for_archive parameter (which defaults to true) can be set to false to have
pg_stop_backup return as soon as the stop backup record is written to the WAL. By default,
pg_stop_backup will wait until all WAL has been archived, which can take some time. This option must be used with caution: if WAL archiving is not monitored correctly then the backup might not include all of the WAL files and will therefore be incomplete and not able to be restored.
The exclusive backup method is deprecated and should be avoided. Prior to PostgreSQL 9.6, this was the only low-level method available, but it is now recommended that all users upgrade their scripts to use non-exclusive backups.
The process for an exclusive backup is mostly the same as for a non-exclusive one, but it differs in a few key steps. This type of backup can only be taken on a primary and does not allow concurrent backups. Moreover, because it creates a backup label file, as described below, it can block automatic restart of the primary server after a crash. On the other hand, the erroneous removal of this file from a backup or standby is a common mistake, which can result in serious data corruption. If it is necessary to use this method, the following steps may be used.
Ensure that WAL archiving is enabled and working.
Connect to the server (it does not matter which database) as a user with rights to run pg_start_backup (superuser, or a user who has been granted EXECUTE on the function) and issue the command:
SELECT pg_start_backup('label');
label is any string you want to use to uniquely identify this backup operation.
pg_start_backup creates a backup label file, called
backup_label, in the cluster directory with information about your backup, including the start time and label string. The function also creates a tablespace map file, called
tablespace_map, in the cluster directory with information about tablespace symbolic links in
pg_tblspc/ if one or more such link is present. Both files are critical to the integrity of the backup, should you need to restore from it.
By default,
pg_start_backup can take a long time to finish. This is because it performs a checkpoint, and the I/O required for the checkpoint will be spread out over a significant period of time, by default half your inter-checkpoint interval (see the configuration parameter checkpoint_completion_target). This is usually what you want, because it minimizes the impact on query processing. If you want to start the backup as soon as possible, use:
SELECT pg_start_backup('label', true);
This forces the checkpoint to be done as quickly as possible.
Perform the backup, using any convenient file-system-backup tool such as tar or cpio (not pg_dump or pg_dumpall). It is neither necessary nor desirable to stop normal operation of the database while you do this. See Section 188.8.131.52 for things to consider during this backup.
As noted above, if the server crashes during the backup it may not be possible to restart until the
backup_label file has been manually deleted from the
PGDATA directory. Note that it is very important to never remove the
backup_label file when restoring a backup, because this will result in corruption. Confusion about when it is appropriate to remove this file is a common cause of data corruption when using this method; be very certain that you remove the file only on an existing primary and never when building a standby or restoring a backup, even if you are building a standby that will subsequently be promoted to a new primary.
Again connect to the database as a user with rights to run pg_stop_backup (superuser, or a user who has been granted EXECUTE on the function), and issue the command:
SELECT pg_stop_backup();
This function terminates backup mode and performs an automatic switch to the next WAL segment. The reason for the switch is to arrange for the last WAL segment written during the backup interval to be ready to archive.
Once the WAL segment files active during the backup are archived, you are done. The file identified by
pg_stop_backup's result is the last segment that is required to form a complete set of backup files. If
archive_mode is enabled,
pg_stop_backup does not return until the last segment has been archived. Archiving of these files happens automatically since you have already configured
archive_command. In most cases this happens quickly, but you are advised to monitor your archive system to ensure there are no delays. If the archive process has fallen behind because of failures of the archive command, it will keep retrying until the archive succeeds and the backup is complete.
When using exclusive backup mode, it is absolutely imperative to ensure that
pg_stop_backup completes successfully at the end of the backup. Even if the backup itself fails, for example due to lack of disk space, failure to call
pg_stop_backup will leave the server in backup mode indefinitely, causing future backups to fail and increasing the risk of a restart failure during the time that
backup_label exists.
Some file system backup tools emit warnings or errors if the files they are trying to copy change while the copy proceeds. When taking a base backup of an active database, this situation is normal and not an error. However, you need to ensure that you can distinguish complaints of this sort from real errors. For example, some versions of rsync return a separate exit code for "vanished source files", and you can write a driver script to accept this exit code as a non-error case. Also, some versions of GNU tar return an error code indistinguishable from a fatal error if a file was truncated while tar was copying it. Fortunately, GNU tar versions 1.16 and later exit with 1 if a file was changed during the backup, and 2 for other errors. With GNU tar version 1.23 and later, you can use the warning options
--warning=no-file-changed --warning=no-file-removed to hide the related warning messages.
Be certain that your backup includes all of the files under the database cluster directory (e.g.,
/usr/local/pgsql/data). If you are using tablespaces that do not reside underneath this directory, be careful to include them as well (and be sure that your backup archives symbolic links as links, otherwise the restore will corrupt your tablespaces).
You should, however, omit from the backup the files within the cluster's
pg_wal/ subdirectory. This slight adjustment is worthwhile because it reduces the risk of mistakes when restoring. This is easy to arrange if
pg_wal/ is a symbolic link pointing to someplace outside the cluster directory, which is a common setup anyway for performance reasons. You might also want to exclude
postmaster.pid and
postmaster.opts, which record information about the running postmaster, not about the postmaster which will eventually use this backup. (These files can confuse pg_ctl.)
It is often a good idea to also omit from the backup the files within the cluster's
pg_replslot/ directory, so that replication slots that exist on the primary do not become part of the backup. Otherwise, the subsequent use of the backup to create a standby may result in indefinite retention of WAL files on the standby, and possibly bloat on the primary if hot standby feedback is enabled, because the clients that are using those replication slots will still be connecting to and updating the slots on the primary, not the standby. Even if the backup is only intended for use in creating a new primary, copying the replication slots isn't expected to be particularly useful, since the contents of those slots will likely be badly out of date by the time the new primary comes on line.
The contents of the directories
pg_stat_tmp/, and
pg_subtrans/ (but not the directories themselves) can be omitted from the backup as they will be initialized on postmaster startup. If stats_temp_directory is set and is under the data directory then the contents of that directory can also be omitted.
Any file or directory beginning with
pgsql_tmp can be omitted from the backup. These files are removed on postmaster start and the directories will be recreated as needed.
pg_internal.init files can be omitted from the backup whenever a file of that name is found. These files contain relation cache data that is always rebuilt when recovering.
The backup label file includes the label string you gave to
pg_start_backup, as well as the time at which
pg_start_backup was run, and the name of the starting WAL file. In case of confusion it is therefore possible to look inside a backup file and determine exactly which backup session the dump file came from. The tablespace map file includes the symbolic link names as they exist in the directory
pg_tblspc/ and the full path of each symbolic link. These files are not merely for your information; their presence and contents are critical to the proper operation of the system's recovery process.
It is also possible to make a backup while the server is stopped. In this case, you obviously cannot use
pg_start_backup or
pg_stop_backup, and you will therefore be left to your own devices to keep track of which backup is which and how far back the associated WAL files go. It is generally better to follow the continuous archiving procedure above.
Okay, the worst has happened and you need to recover from your backup. Here is the procedure:
Stop the server, if it's running.
If you have the space to do so, copy the whole cluster data directory and any tablespaces to a temporary location in case you need them later. Note that this precaution will require that you have enough free space on your system to hold two copies of your existing database. If you do not have enough space, you should at least save the contents of the cluster's
pg_wal subdirectory, as it might contain logs which were not archived before the system went down.
Remove all existing files and subdirectories under the cluster data directory and under the root directories of any tablespaces you are using.
Restore the database files from your file system backup. Be sure that they are restored with the right ownership (the database system user, not
root!) and with the right permissions. If you are using tablespaces, you should verify that the symbolic links in
pg_tblspc/ were correctly restored.
Remove any files present in
pg_wal/; these came from the file system backup and are therefore probably obsolete rather than current. If you didn't archive
pg_wal/ at all, then recreate it with proper permissions, being careful to ensure that you re-establish it as a symbolic link if you had it set up that way before.
If you have unarchived WAL segment files that you saved in step 2, copy them into
pg_wal/. (It is best to copy them, not move them, so you still have the unmodified files if a problem occurs and you have to start over.)
Set recovery configuration settings in
postgresql.conf (see Section 20.5.4) and create a file
recovery.signal in the cluster data directory. You might also want to temporarily modify
pg_hba.conf to prevent ordinary users from connecting until you are sure the recovery was successful.
Start the server. The server will go into recovery mode and proceed to read through the archived WAL files it needs. Should the recovery be terminated because of an external error, the server can simply be restarted and it will continue recovery. Upon completion of the recovery process, the server will remove
recovery.signal (to prevent accidentally re-entering recovery mode later) and then commence normal database operations.
Inspect the contents of the database to ensure you have recovered to the desired state. If not, return to step 1. If all is well, allow your users to connect by restoring
pg_hba.conf to normal.
The key part of all this is to set up a recovery configuration that describes how you want to recover and how far the recovery should run. The one thing that you absolutely must specify is the
restore_command, which tells PostgreSQL how to retrieve archived WAL file segments. Like the
archive_command, this is a shell command string. It can contain
%f, which is replaced by the name of the desired log file, and
%p, which is replaced by the path name to copy the log file to. (The path name is relative to the current working directory, i.e., the cluster's data directory.) Write
%% if you need to embed an actual
% character in the command. The simplest useful command is something like:
restore_command = 'cp /mnt/server/archivedir/%f %p'
which will copy previously archived WAL segments from the directory
/mnt/server/archivedir. Of course, you can use something much more complicated, perhaps even a shell script that requests the operator to mount an appropriate tape.
It is important that the command return nonzero exit status on failure. The command will be called requesting files that are not present in the archive; it must return nonzero when so asked. This is not an error condition. An exception is that if the command was terminated by a signal (other than SIGTERM, which is used as part of a database server shutdown) or an error by the shell (such as command not found), then recovery will abort and the server will not start up.
Not all of the requested files will be WAL segment files; you should also expect requests for files with a suffix of
.history. Also be aware that the base name of the
%p path will be different from
%f; do not expect them to be interchangeable.
WAL segments that cannot be found in the archive will be sought in
pg_wal/; this allows use of recent un-archived segments. However, segments that are available from the archive will be used in preference to files in
Normally, recovery will proceed through all available WAL segments, thereby restoring the database to the current point in time (or as close as possible given the available WAL segments). Therefore, a normal recovery will end with a "file not found" message, the exact text of the error message depending upon your choice of
restore_command. You may also see an error message at the start of recovery for a file named something like
00000001.history. This is also normal and does not indicate a problem in simple recovery situations; see Section 26.3.5 for discussion.
If you want to recover to some previous point in time (say, right before the junior DBA dropped your main transaction table), just specify the required stopping point. You can specify the stop point, known as the "recovery target", either by date/time, named restore point or by completion of a specific transaction ID. As of this writing only the date/time and named restore point options are very usable, since there are no tools to help you identify with any accuracy which transaction ID to use.
The stop point must be after the ending time of the base backup, i.e., the end time of
pg_stop_backup. You cannot use a base backup to recover to a time when that backup was in progress. (To recover to such a time, you must go back to your previous base backup and roll forward from there.)
If recovery finds corrupted WAL data, recovery will halt at that point and the server will not start. In such a case the recovery process could be re-run from the beginning, specifying a "recovery target" before the point of corruption so that recovery can complete normally. If recovery fails for an external reason, such as a system crash or if the WAL archive has become inaccessible, then the recovery can simply be restarted and it will restart almost from where it failed. Recovery restart works much like checkpointing in normal operation: the server periodically forces all its state to disk, and then updates the
pg_control file to indicate that the already-processed WAL data need not be scanned again.
The ability to restore the database to a previous point in time creates some complexities that are akin to science-fiction stories about time travel and parallel universes. For example, in the original history of the database, suppose you dropped a critical table at 5:15PM on Tuesday evening, but didn't realize your mistake until Wednesday noon. Unfazed, you get out your backup, restore to the point-in-time 5:14PM Tuesday evening, and are up and running. In this history of the database universe, you never dropped the table. But suppose you later realize this wasn't such a great idea, and would like to return to sometime Wednesday morning in the original history. You won't be able to if, while your database was up-and-running, it overwrote some of the WAL segment files that led up to the time you now wish you could get back to. Thus, to avoid this, you need to distinguish the series of WAL records generated after you've done a point-in-time recovery from those that were generated in the original database history.
To deal with this problem, PostgreSQL has a notion of timelines. Whenever an archive recovery completes, a new timeline is created to identify the series of WAL records generated after that recovery. The timeline ID number is part of WAL segment file names so a new timeline does not overwrite the WAL data generated by previous timelines. It is in fact possible to archive many different timelines. While that might seem like a useless feature, it's often a lifesaver. Consider the situation where you aren't quite sure what point-in-time to recover to, and so have to do several point-in-time recoveries by trial and error until you find the best place to branch off from the old history. Without timelines this process would soon generate an unmanageable mess. With timelines, you can recover to any prior state, including states in timeline branches that you abandoned earlier.
Every time a new timeline is created, PostgreSQL creates a "timeline history" file that shows which timeline it branched off from and when. These history files are necessary to allow the system to pick the right WAL segment files when recovering from an archive that contains multiple timelines. Therefore, they are archived into the WAL archive area just like WAL segment files. The history files are just small text files, so it's cheap and appropriate to keep them around indefinitely (unlike the segment files which are large). You can, if you like, add comments to a history file to record your own notes about how and why this particular timeline was created. Such comments will be especially valuable when you have a thicket of different timelines as a result of experimentation.
The default behavior of recovery is to recover to the latest timeline found in the archive. If you wish to recover to the timeline that was current when the base backup was taken or into a specific child timeline (that is, you want to return to some state that was itself generated after a recovery attempt), you need to specify
current or the target timeline ID in recovery_target_timeline. You cannot recover into timelines that branched off earlier than the base backup.
Some tips for configuring continuous archiving are given here.
It is possible to use PostgreSQL's backup facilities to produce standalone hot backups. These are backups that cannot be used for point-in-time recovery, yet are typically much faster to backup and restore than pg_dump dumps. (They are also much larger than pg_dump dumps, so in some cases the speed advantage might be negated.)
As with base backups, the easiest way to produce a standalone hot backup is to use the pg_basebackup tool. If you include the
-X parameter when calling it, all the write-ahead log required to use the backup will be included in the backup automatically, and no special action is required to restore the backup.
If more flexibility in copying the backup files is needed, a lower level process can be used for standalone hot backups as well. To prepare for low level standalone hot backups, make sure
wal_level is set to
replica or higher,
archive_mode to
on, and set up an
archive_command that performs archiving only when a switch file exists. For example:
archive_command = 'test ! -f /var/lib/pgsql/backup_in_progress || (test ! -f /var/lib/pgsql/archive/%f && cp %p /var/lib/pgsql/archive/%f)'
This command will perform archiving when
/var/lib/pgsql/backup_in_progress exists, and otherwise silently return zero exit status (allowing PostgreSQL to recycle the unwanted WAL file).
With this preparation, a backup can be taken using a script like the following:
touch /var/lib/pgsql/backup_in_progress psql -c "select pg_start_backup('hot_backup');" tar -cf /var/lib/pgsql/backup.tar /var/lib/pgsql/data/ psql -c "select pg_stop_backup();" rm /var/lib/pgsql/backup_in_progress tar -rf /var/lib/pgsql/backup.tar /var/lib/pgsql/archive/
The switch file
/var/lib/pgsql/backup_in_progress is created first, enabling archiving of completed WAL files to occur. After the backup the switch file is removed. Archived WAL files are then added to the backup so that both base backup and all required WAL files are part of the same tar file. Please remember to add error handling to your backup scripts.
If archive storage size is a concern, you can use gzip to compress the archive files:
archive_command = 'gzip < %p > /mnt/server/archivedir/%f.gz'
You will then need to use gunzip during recovery:
restore_command = 'gunzip < /mnt/server/archivedir/%f.gz > %p'
archive_command Scripts
Many people choose to use scripts to define their
archive_command, so that their
postgresql.conf entry looks very simple:
archive_command = 'local_backup_script.sh "%p" "%f"'
Using a separate script file is advisable any time you want to use more than a single command in the archiving process. This allows all complexity to be managed within the script, which can be written in a popular scripting language such as bash or perl.
Examples of requirements that might be solved within a script include:
Copying data to secure off-site data storage
Batching WAL files so that they are transferred every three hours, rather than one at a time
Interfacing with other backup and recovery software
Interfacing with monitoring software to report errors
When using an
archive_command script, it's desirable to enable logging_collector. Any messages written to stderr from the script will then appear in the database server log, allowing complex configurations to be diagnosed easily if they fail.
At this writing, there are several limitations of the continuous archiving technique. These will probably be fixed in future releases:
If a
CREATE DATABASE command is executed while a base backup is being taken, and then the template database that the
CREATE DATABASE copied is modified while the base backup is still in progress, it is possible that recovery will cause those modifications to be propagated into the created database as well. This is of course undesirable. To avoid this risk, it is best not to modify any template databases while taking a base backup.
CREATE TABLESPACE commands are WAL-logged with the literal absolute path, and will therefore be replayed as tablespace creations with the same absolute path. This might be undesirable if the log is being replayed on a different machine. It can be dangerous even if the log is being replayed on the same machine, but into a new data directory: the replay will still overwrite the contents of the original tablespace. To avoid potential gotchas of this sort, the best practice is to take a new base backup after creating or dropping tablespaces.
It should also be noted that the default WAL format is fairly bulky since it includes many disk page snapshots. These page snapshots are designed to support crash recovery, since we might need to fix partially-written disk pages. Depending on your system hardware and software, the risk of partial writes might be small enough to ignore, in which case you can significantly reduce the total volume of archived logs by turning off page snapshots using the full_page_writes parameter. (Read the notes and warnings in Chapter 30 before you do so.) Turning off page snapshots does not prevent use of the logs for PITR operations. An area for future development is to compress archived WAL data by removing unnecessary page copies even when
full_page_writes is on. In the meantime, administrators might wish to reduce the number of page snapshots included in WAL by increasing the checkpoint interval parameters as much as feasible.
If you see anything in the documentation that is not correct, does not match your experience with the particular feature or requires further clarification, please use this form to report a documentation issue.

subset: commoncrawl


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text:
From 2012.igem.org
Realizing Flower Fairy in real world
Have you ever seen flower fairies? Probably the answer is, "NO" (though some of you might have come across them in your childhood), because they are imaginary creatures which exist only in fairy tales. Don't you think it would be wonderful if you could live with flower fairies? In addition to the happy feelings, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!
In order to realize our dream, we focused on FT protein, known as Florigen.This protein is a kind of plant hormone. First, FT protein is produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.
Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!
When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT protein is secreted and penetrate the cell membrane of a plant, and the plant starts blooming.
We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.
These four steps are "EXPRESSION","SECRETION", "PENETRATION", and "ACTIVATION"
In each step, we have some problems to be attacked.
"EXPRESSION"; It is unclear whether E.coli (prokaryote) can express FT protein, because FT protein is derived from plant cells (eukaryote).
"SECRETION"; After produced, FT protein has to get out of the E.coli.
"PENETRATION"; FT protein has to penetrate into plant cells.
"ACTIVATION"; Even if FT protein could get into the cells, it is not clear whether FT protein produced by E.coli can activate genes in shoot apex cells and induce flower formation.
The first step is EXPRESSION.
We had to make E.coli produce FT protein.
However, in the natural environment, wildE.coli don't have FT gene. Therefore we tried to make a new BioBrick part including FT gene and introduce it into E.coli.
Modifying FT gene for Biobrick
FT gene is derived from Arabidopsis thaliana, a model plant. Professor Araki in Kyoto University kindly gave us FT cDNA in TOPO blunt end 2(Invitrogen). FT cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified FT gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to FT. Finally, we could make new biobrick parts of FT (Fig.1-1 C).
Confirming expression of FT
We constructed the plasmid shown in the Fig.1-2. One was FT gene with T7 promoter (BBa_I719005) and 6His tag, and the other was FT gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of E.coli, the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from E.coli using affinity chromatography.
We needed to confirm that E. coli can express FT protein, because there was a possibility that E.coli can not translate FT or FT is unstable or toxic in E.coli.
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).
So we successfuly confirmed the FT expression!
We succeeded in confirming the mutation and the expression of FT and 6 His:FT protein in E.coli!
The second step is SECRETION. We want E.coli to secrete FT protein to outside of the cell.
Now our E.coli can produce FT protein, but a big issue still remains: how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables E.coli to transport FT protein continuously.
Proteins are required to go through two membranes
E.coli have two membranes: inner membrane and outer membrane. To transport FT protein to outside of E.coli, therefore, FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes.
TorA signal enables proteins to go through the inner membrane via tat pathway
Wild E.coli have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation(Tat) pathway. Tat pathway is more suitable than other secretion systems for us, because by Tat pathway E.coli secrete proteins into the periplasm keeping proteins' conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and transports into the periplasm only proteins that have a TorA signal at their N terminals.
We improved usability of TorA signal biobrick
We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as BBa_K638402) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS, which regulate additional processing time for iGEMers who use the parts. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, therefore, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, but TorA would be expressed.
To solve these problems, we produced new applicable TorA signal BBa_K797002 (Fig.2-2). Our part has two improvements. BBa_K797002 contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced BBa_K797002 and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly.
Kil protein enables proteins to go through outer membrane
With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of E.coli. So in our project, we introduced kil gene into E.coli. But the function of outer membrane is essential for E.coli to survive. Overexpression of kil gene, therefore, causes cell death. For this reason, we must check whether kil gene is harmful or not under our condition.
Kil protein had no significant effect on E.coli's growth
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of E.coli. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of E.coli.
We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make E.coli to secrete FT protein without cell lysis!!(Fig.2-7)
Improvement of secretion system
Now we can enable E.coli to secrete FT proteins to the outside by using Tat pathway and kil protein. But E.coli secrete only a small amount of FT proteins when they use Tat transporters which they inherently have. To make E.coli secrete enough amount of FT proteins, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild E.coli have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H+ concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion. We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make E.coli secrete more proteins with TorA.(Fig.2-9)
Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC (BBa_K797000) and the sequence of pspA (BBa_K797001) individually, and then, we made Tat construction composed of constitutive promoter (BBa_J23107), TatABC (BBa_K797000), pspA (BBa_K797001) and double terminator (BBa_B0015). We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.
The third step is PENETRATION. In order to induce flower formation, FT proteins from E.coli must enter into plant cells. This is because FT proteins upregulate other proteins which lead to flower formation in plant cells.
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.
Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.
R9 peptide enables FT protein penetrate membranes by endocytosis
R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.
This is the mechanism of how R9 peptides work(Fig3-2).
Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.
Secondly, the cell responds to this stimulus and starts to endocytose.
Finally, proteins around the endocytosing region of the cell is taken into the cell.
R9 peptides seem to work effectively regardless of whether or not R9 peptides are fused with target proteins. However, there are few examples about endocytosis of plants, so we needed to check the function of R9 when used on plant cells.
From the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not.
Considering this system, target proteins near the R9 peptides are taken by endocytosis.
In other words, it can be said that it induces endocytosis regardless of whichever R9 peptides and target proteins are fused.
We tried to wonder whether we should fuse R9 and target proteins.
From this system, we thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. Therefore, we considered that fusing R9 peptide with a target protein may leads to higher efficiency.
So, we tried to fuse R9 peptides with target proteins to increase penetration efficiency
We transformed E.coli, arranging R9's sequence and target protein's gene in line. And after that we tried to make E.coli express R9::GFP fusion protein.
In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, E.coli expressing the R9::GFP fusion protein was poor in growth(Fig3-3). Then, we checked R9's effect on R9::GFP fusion protein's expression by Western blotting and RT-PCR. We succeeded in confirming the existence of the mRNA (Fig3-4), but we did not find the protein (Fig3-5). These results indicated poor translation or quick breakdown of the protein.
We used the existing GFP generator part, BBa_I746915.
Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein.
We used R9 and GFP separately and to check the R9 peptides function
We wondered whether R9 peptides work properly and put FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell's endocytosis. We, therefore, need to verify the function of R9 peptides. For this reason, we performed following experiment(Fig3-6).
First, we removed the cuticle of Arabidopsis thaliana's leaves by scratching them. Second, we soaked the leaves into a solution of only GFP, or GFP and R9 for each. After 5 minutes we washed cells by PBS in order to put GFP and R9 peptides away from leaves. After that, we compared leaves having soaked in only GFP and ones having soaked R9 and GFP. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).
The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or around plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.
We can make FT protein penetrate cell membrane of plants by R9 peptide function!.
4. Activation
On the final step; Activation. We verified whether FT protein made in E.coli worked normally in plant cells.
Actually, FT protein is a transcriptional factor so that we can check the ability of FT protein by observing the transcript levels of genes FT protein upregulate.
The detail function of FT as a transcriptional factor
FT protein increases transcript activity of several genes which leads to flower formation. The protein can .
Injecting FT and verifying its function by RT-PCR
To evaluate the function of FT which our Fairy made, we used leaf cells of Arabidopsis thaliana , not soot apex. Plants produce FT protein in their leaves and carry it to soot apex. For these reason, we assumed that our Flower Fairy can make flowers bloom only if it can introduce active FT protein in leaves. In addition to that, we can get lots of sample easily. Previous research said that FT gene can also up-regulate some gene,such as FUL and SEP3, in leaves so that We injected FT protein into leaves and conducted RT-PCR to value the amount of expressed RNA.
FT protein is injected by a syringe. Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control. GFP was used as a control to confirm the result of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)
We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.
Fig.4-2 is the result of RT-PCR.
As shown above, we got no correct ampicon band, even of tubulin, which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.
As shown in the Fig.4-3, RNA samples in this time are degradated.
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)
Establishing an effective way of RNA purification
We found that RNA purification method should be improved for performing RT-PCR successfully .
We improved following things;
1. Total leaf volume was increased.
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.
3. We centrifuged samples and collected supernatant twice after adding ISOGEN.
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.)
Then we used better quality of RNA for reverse transcription and retried RT-PCR. Fig.4-6 shows the result of this RT-PCR.
Conducting RT-PCR and qPCR again by using new RNA purification
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL
Checking whether FT protein really introduced or not
- Mutate FT sequence
- Standardize FT as an iGEM part
- Confirm expression of FT protein in E.coli
- Modify TorA signal to be easy to use the signal more
- Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator
- Standardize kil gene
- Multiply TatABC in order to strengthen Tat secretion system--not yet
- Keep GFP in or around plant cells using R9 peptide
- Introduce FT in plant cells using R9 peptide--not yet
- Get high quality of RNA
- Amplify genes successfully
- Check the function of FT--not yet
In order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It's cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn't enter plant cells. If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.
If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.
When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.
When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.
If the cause was that FT didn't enter plant cell or FT was decomposed in the cell, FT will not be confirmed. When FT didn't enter plant cell, we will reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s. Wnen FT was decomposed in the cell, the problem may be solved by fusing R9 with FT's C-terminal, not N-terminal, because there are some references mentioning that protein tagged with R9 at it's N-terminal may be decomposed in the cell.
Future Works
We noticed only flowering and florigen in this time but there are many other plant hormones. We made translocation pathway from E.coli into plant cells, so we will be able to introduce plant hormones into plant cells if E.coli can make them. It means we can control plant growth in any stage through genetically engineered E.coli. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.
Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to control all living cells using this technology.
We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.
[1]Microsugar Chang et al.(2005) "Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells" Plant Cell Physiol, 46(3), 482–488
[2]Paula Teper-Bamnolker and Alon Samach1.(2005) "The flowering integrator FT regulates SEPALLATA3 and FRUITFULL accumulation in Arabidopsis leaves" The Plant Cell, 17, 2661–2675
[3]Philip A. Wigge et al.(2005) "Integration of spatial and temporal information during floral induction in Arabidopsis" Science, 309(5737), 1056-1059
[4]Sara Trabulo et al.(2010) "Cell-penetrating peptides—mechanisms of cellular uptake and generation of delivery systems" Pharmaceuticals, 3, 961-993
[5]Unnamalai N, Kang BG, Lee.(2004) "Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells" FEBS Lett 21, 566(1-3), 307-10
[6]Tracy Palmer and Ben C. Berks.(2012) "The twin-arginine translocation (Tat) protein export pathway" Nat Rev Microbiol, 10(7), 483-96
[7]Choi JH, Lee SY.(2004) "Secretory and extracellular production of recombinant proteins using Escherichia coli" Appl Microbiol Biotechnol, 64(5), 625-35
[8]Miksch G, Fiedler E, Dobrowolski P, Friehs K.(1997) "The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase" Arch Microbiol, 167(2-3), 143-50
[9]Seibel BA, Walsh PJ.(2002) "Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage" J Exp Biol, 205(Pt 3), 297-306
[10]Thomas JD, Daniel RA, Errington J, Robinson C.(2001) "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli" Mol Microbiol, 39(1), 47-53
[11]Suit JL, Luria SE.(1988) "Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death" J Bacteriol, 170(10), 4963-4966
[12]DeLisa MP, Lee P, Palmer T, Georgiou G.(2004) "Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway" J Bacteriol, 186(2), 366-373
[13]Araki, T et al.(2005) "FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex" Science 309(5737), 1052–1056


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