IGEM:Caltech/2008/Project/Lactose intolerance

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iGEM 2008

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Lactose Summary

We want to make two plasmids:
Plasmid #1 - A constitutive promoter with LacZ and LacY.
The general idea is to overexpress LacY (Lactose transporter) with LacZ (Beta-Galactosidase). When lactose is present, the cell will uptake lactose and will flip on the Lac promoter repressed by lacI when enough lactose is present. We then express lysozyme and holin which will lyse the cell releasing beta-gal cleaving lactose into glucose and galactose. Glucose and galactose will then be taken up by the host in the colon.

Lactose Outline

Low expression - J23113
Medium expression - J23106
High expression - J23100
After LacZ culture is grown, mini prep DNA.
Set up o/n cultures.
1. LacZ in Amp
2. pSB2K3 in Km
3. p1005 in Amp
Mini prep'd LacZ and pSB2K3, eluted in 50 μL.
LacZ conc. = 115.4 ng/μL
pSB2K3 conc. = 427.4 ng/μL
Rxn setup:
Set up digests for LacZ and LacY.
1. Cut LacY XP
2. Cut RBS34 SP
3. Cut RBS32 SP
4. Cut High Copy Plasmid XP
5. Cut LacZ ES
6. Cut TT EX
Ran another LacY PCR with template from previous LacY purification.
Treated digest backbones with CIP, and heat inactivated.
B0031: 113 ng/ul
B0033: 120 ng/ul
Ran last nights digests on a gel and gel extracted them.
Set up 6 ligations and transformed them.
1. LacY + RBS34
2. LacY + RBS32
3. LacY + pSB1A2
4. Control: RBS34
5. Control: RBS32
6. Control: pSB1A2
To Do List:
1. RBS LacZ - Already have
3. B0031/33 (Weak RBS's) - Cut Sun
5. pSB1A2 p1010(ccdb) - cut Sun
Transformed B0015 Terminator.
PCR purified LacY
Set up digests of:
1. B0015 EX
2. B0031 SP
3. B0033 SP
4. B0032-LacY ES
5. LacY XP
6. pSB1A2-LacY ES
Gel extracted digests, set up ligations and transformed for:
1. LacZ + B0015
2. RBS32-LacY + B0015
3. LacY + B0031
4. LacY + B0033
5. pSB1A2-LacY + B0015
6. CONTROL B0015
7. CONTROL B0031
8. CONTROL B0033
Lysis cassette primers arrived.
Transformations looked good. We used the less competent cells from upstairs instead of D10HB, so I decided to spin down and plate all cells. All plates had colonies, background from controls were less than ligations, although B0031 had about the same background. Set up colony PCR's and ran on a gel.
Colony PCR's FAILED! Time to start over.
The Plan for now since we have LacY in pSB1A2:
1. LacY --> B0031/33/15
2. LacY --> SDM and primer design
3. LacZ --> B0015
4. Lysis cassette --> 1A2
PCR'd the lysis cassette out of Lambda Zap. Two bands showed, one at 1KB and 1 at about 1.3KB. The band at 1.3KB was much darker and that is what we expected our size to be.
Happy Fourth of July!
PowerPoint for 7/9/08
iGEM Meeting #3
The past few days have been failure!
Tried to re-clone LacY into B0015, B0031/33 and LacZ into B0015. It failed, and re-did the digests tonight. The analytical digest looked good, so hopefully we can gel extract clean bands. We are also working on the lysis cassette to place into pSB1A2 which I ligated and transformed tonight. Hopefully there will be little background from the control. If that looks good, we will screen with colony PCR tomorrow.
Transformation of Lysis Cassette + pSB1A2 looked beautiful. The control plate had 0 colonies while the ligation had a fair amount. Ran a colony PCR of 9 different colonies + a control of 1A2 prep. There were 4 bands at about 1.5KB (what we expect since lysis = 1.3 + VF VR = .2) and 3 bands had nothing. 1 band was VERY flooded, so something may have gone weird in that lane. We hopefully reached SUCCESS!
Will grow o/n cultures of two colonies that looked good and will send in for sequencing on Monday morning.
The digest of all backbones were gel extracted and the ligations will be carried out today.
LacY into 31/33, and LacZ into 15, for a total of 3 reactions and 3 controls.
Sucess!! The ligations had about a couple hundred fold (if not more) over the control plates. I will screen with colony PCR today and if they look good, I'll send them in for sequencing along with the lysis cassette and SDM LacY.
Colony PCR's looked beautiful! Will grow 2 cultures o/n and send 1 in for sequencing.
Sequencing for Monday:
1. SDM LacY (2 colonies)
2. Lysis Cassette + 1A2 (2 colonies)
3. LacY + 31/33 (2 colonies each)
4. LacZ + 15 (2 colonies)
Sequencing preps have been set up and will be delivered Monday morning
I wait for sequencing... Colony PCR's on SDM LacY + H looked good, will prep and sequence tomorrow. Made lots plates for Allen =)
Will update soon... For now, keep yourself entertained with my glorious PowerPoint.
iGEM Meeting #4
So its been a while since I updated... but here's the good news. We now have the following constructs (will be confirmed by colony PCR in 4 hours! QQ)
1. Lysis + b0015 (2K3)
2. 31 LacY + b0015 (1AK3)
3. 33 LacY + b0015 (1AK3)
4. 34 LacZ b0015 + Str. Po. (J23100)
If these work by sequencing, we can put together the final round of constructs and begin our assays:
1. Lysis b0015 + A81 (Lac Ind. Po.) in 2K3
2. Lysis b0015 + B4 (Lac Ind. Po.) in 2K3
3. 31 LacY b0015 + Weak Const. Po in 1AK3
4. 31 LacY b0015 + Med Const. Po in 1AK3
5. 31 LacY b0015 + Str Const. Po in 1AK3
6. 33 LacY b0015 + Weak Const. Po in 1AK3
7. 33 LacY b0015 + Med Const. Po in 1AK3
8. 33 LacY b0015 + Str Const. Po in 1AK3
9. 34 LacZ b0015 Str. Po (we will hopefully have the assay results)
10. RBS GFP TT + A81 (To test if our Lac Ind. Po. is working)
11. RBS GFP TT + B4 (To test if our Lac Ind. Po. is working)
Lots of stuff is working. =) More details later! SUCCESS!
Three cloning steps away from success.

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