Team:MIT/Notebook/

From 2011.igem.org

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<p>LR Reactions<br/>
<p>LR Reactions<br/>
Utilizing protocol here: <a href="/confluence/display/iGEM2011/LR+Protocol" title="LR Protocol">LR Protocol</a></p>
Utilizing protocol here: <a href="/confluence/display/iGEM2011/LR+Protocol" title="LR Protocol">LR Protocol</a></p>
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Revision as of 20:04, 28 September 2011

Navigation

  • Overview
  • Week 1
  • Week 2
  • Week 3
  • Week 4
  • Week 5
  • Week 6
  • Week 7
  • Week 8
  • Week 9
  • Week 10
  • Week 11
  • Week 12

Overview

Here you can see what we did weekly in the lab!

Week 1: June 6 - June 11

In the first week we began constructing basic reporter DNA constructs that have various combinations of promoters and genes. The mix-and-matching of promoters and genes was done using the LR reaction following the Gateway protocol. Our promoters included TRE, minCMV, Hef1a, Hefl1a-LacO, and our genes included eYFP, mKate, and eBFP2. Transformed and inoculated colonies from the LR reactions and performed restriction digests. Approximately half of the LR reactions were successful and became DNA parts that we could use later on.

June 7, 2011

Date
Assignee
DEST_R4R2
L4-R1 Promoter
L1-L2
Gene
LR?
Tube Number
Colony Number,
Antibiotic, Person 
Protocol Used
6.7.2011
Michelle
Louis
2-3
Tre
C1434VP16
Yes. 6.7.2011, 5pm
11
5, AMP, KH
Standard, 30C for 16h
6.7.2011
Michelle
Louis
2-3
Tre
Mnt1VP16
Yes. 6.7.2011, 5pm
12
4, AMP, KH Standard, 30C for 16h
6.7.2011
Tyler
2-3
Tre
LacIKrab
Yes. 6.7.2011, 5pm
6
30, AMP, KH Standard, 30C for 16h
6.7.2011
Jenny
Divya
3-4
minCMV-7xMnt1
eYFP
Yes. 6.7.2011, 5pm
9
0, AMP, KH, needs to be redone
Standard, 30C for 16h
6.7.2011
Jenny
Divya
2-3
Tre
LexAVP16
Yes. 6.7.2011, 5pm
10
2, AMP, KH, needs to be redone
Standard, 30C for 16h
6.7.2011 Mariola
Kenneth
3-4
minCMV 1xC1434
eYFP
Yes. 6.7.2011, 5pm
7
3, AMP, KH, needs to be redone Standard, 30C for 16h
6.7.2011 Mariola
Kenneth
3-4 minCMV 4xLexA
eYFP
Yes. 6.7.2011, 5pm
8
0, AMP, KH, needs to be redone
Standard, 30C for 16h

LR Reactions
Utilizing protocol here: LR Protocol

Date
Assignee
DEST_R4R2
L4-R1 Promoter
L1-L2
Gene
LR?
Tube Number
Colony Number,
Antibiotic, Person 
Protocol Used
6.9.2011
Grant
3-4
minCMV 4xLexA eYFP Yes. 6.9.2011, 3pm
12
10, AMP, GR  
Standard + Grown at 37C in LB not SOC.
6.9.2011
Jon C
3-4
minCMV-4xMnt1
eYFP
Yes. 6.9.2011, 3:45pm 2
13, AMP, GR
Standard + Grown at 37C in LB not SOC.
6.9.2011
Jon C
3-4
minCMV-4xCI434
eYFP
Yes. 6.9.2011, 3:45pm 1
7, AMP, GR Standard + Grown at 37C in LB not SOC.
6.9.2011
Jon C
3-4
Hef1a-LacO
eYFP
Yes, 6.9.2011, 4:20pm
5
25, AMP, GR
Standard + Grown at 37C in LB not SOC.
6.9.2011
Semon
4-5
Tre
mKate
Yes, 6.9.2011 4:27pm
3
6, AMP, GR
Standard + Grown at 37C in LB not SOC.
6.9.2011
Semon
1-2
Hef1a
eBFP2
Yes, 6.9.2011, 4.27pm
4
3, AMP, GR
Standard + Grown at 37C in LB not SOC.


Transformation

Date
Assignee
DEST_R4R2
L4-R1 Promoter
L1-L2
Gene
LR?
  Colony Number,
Antibiotic
Protocol Used
6.9.2011
Mariola


eYFP
no. Replication stock.
 
2, AMP
Transformation (~ 2 hr 20 min) using LB instead of SOC

Miniprep

Date Assignee
DNA
Quantity
Time collected
6.9.2011
Divya-Jenny
pEXPR_2-3_Tre:LexAVP16
52.2 ng/uL
12pm
6.9.2011
Kenneth
pEXPR_3-4_minCMV-CI434:eYFP (A)
70 ng/ul
12pm
6.9.2011
Kenneth pEXPR_3-4_minCMV-CI434:eYFP (B)
174 ng/uL
12pm
6.9.2011
Louis
pEXPR_2-3_Tre:C1434VP16
234.6 ng/uL
12pm
6.9.2011
Tyler
pEXPR_2-3_Tre:Lac/Krab
130.7 ng/uL
12pm
6.9.2011
Michelle
pEXPR_2-3_Tre:Mnt1VP16
110.0 ng/uL
12pm
6.9.2011
Michelle
pDEST_2-3_ccdB
117.6 ng/uL
12pm

Restriction Digests

Assignee
DNA
Enzyme
Expected Results
Picture of Gel
Time Incubated Comments
Louis
pEXPR_2-3_Tre:C1434VP16 NdeI
6700 bp
800 bp
a.3 6/9/11
3:50 PM

   
Kenneth
pEXPR_3-4_minCMV-CI434:eYFP (A+B)
NcoI and SacII
2450 bp
4650 bp
A: a.6
B: a.7
6/9/11
3:00 PM
A did not cut as expected. B looks good.
Digestion Mix: 2 uL NEB4, 0.5 uL of each enzyme, A: 7.1 uL/B: 2.9 uL of DNA, 10.4 uL/14.6 uL of H20
   
Michelle
pEXPR_2-3_Tre:Mnt1VP16
BglI
3700 bp
2200 bp
1300 bp
a.1 6/9/11
4:00PM
DNA appeared to be uncut. Need to redo, possibly with a new enzyme and more DNA.
   
Michelle
pDEST_2-3_ccdB
NcoI and NheI
6100 bp
 1700 bp
a.2 6/9/2011
4:25 PM
Attained bands at correct positions. Other band represents partially cut DNA in double digest.
   
Divya
pEXPR_2-3_Tre:LexAVP16
HincII
  a.4 6/9/2011
4:45 PM
- Bands didn't show up. Needs to be redone.
- DNA may have degraded. Need to redo Nanodrop as well.
   
Tyler pEXPR_2-3_Tre:Lac/Krab SalI 3 bands:
5288 bp
1510 bp
1241 bp

cuts gene
a.5 6/9/2011
3:00 PM
Digestion:
4 uL * 130.7 ng/uL = 522.8 ng DNA
2 uL NEB3
2 uL BSA
11 uL H2O
1 uL SalI

Total: 20 uL

Only saw one band at 3500 bp
Needs to be redone possibly with another restriction enzyme
   

Gels

Label Picture
a

June 10, 2011

Restriction Gels

Assignee DNA
Enzyme
Expected result
Time Incubated
Comments
Louis
pEXPR_2-3_Tre:C1434VP16 EcoRI (Buffer 2)
5500 bp
1050 bp
650 bp
360 bp
6/10/11
11:15 AM
 
Michelle
pEXPR_2-3_TRE:Mnt1VP16 (A)
NcoI and NheI
5700 bp
1300 bp
6/10/11
11:40 AM
Digestion:
6 uL DNA
1 uL NcoI
1 uL Nhe1
2 uL NEB2
2 uL BSA
8 uL H2O

Failed.
Michelle
pEXPR_2-3_TRE:Mnt1VP16 (B)
NheI and SphI
1700 bp
5300 bp
6/10/11
11:40 AM
Digestion:
6 uL DNA
1 uL NheI
1 uL SphI
2 uL NEB2
2 uL BSA
8 uL H2O

Failed. Redoing LR on Monday June 13th.
Tyler pEXPR_2-3_Tre:Lac/Krab SalI 3 bands:
5288 bp
1510 bp
1241 bp

cuts gene
6/10/2011
3:00 PM
Digestion: 4 uL * 130.7 ng/uL = 522.8 ng DNA
2 uL NEB3
2 uL BSA
11 uL H2O
1 uL SalI
Total: 20 uL

***Worked (see gel below - b.1)
Stored in -80C freezer

Comments:

pEXPR_2-3_Tre:LexAVP16 and pEXPR_3-4_minCMV-7xMnt1:eYFP are being entirely redone.

pEXPR_2-3_Tre:LexAVP16 (post-miniprep) was lost. pEXPR_3-4_minCMV-7xMnt1:eYFP LR was unsuccessful (no bacteria grew).

LR and transformation will be done on Saturday. Miniprep and restriction mapping will be done on Sunday

Label Gel
Legend
b

Column 0: HyperLadder
Column 1: pEXPR_2-3_Tre:Lac/Krab
Column 4: pEXPR_2-3_Tre:C1434VP16
Column 6: pEXPR_2-3_TRE:Mnt1VP16 (A)
Column 7: pEXPR_2-3_TRE:Mnt1VP16 (B)

June 11, 2011

Set of LRs from 6/9 (GR, JC, and SR) cell counts taken; inoculated by GR/JC. 3 cells taken per plate.

Assignee
DNA
Enzyme
Expected Results
Time Incubated
Comments
Sam
pEXPR_1-2_Hef:eBFP
NcoI & ApaL1
3750bp, 3180bp, 1250bp 45 min If it doesn't work there should be a whole mess of fragments (including a small 600bp one)
1mL each enz; 2 mL Buf; 1 mL BSA; 2mL DNA; 13mL H2O (Standard Protocol with extra BSA)
Sam
pEXPR_4-5_Tre:mKate
Bgl1
1270bp, 2630bp, 3380bp 45 min 1mL enz; 2mL DNA; 2mL BSA; 2mL Buf; 15mL H2O (Standard Protocol with extra BSA and water - not quite 10x)

Week 2: June 12 - June 18

In the second week we attempted midipreps instead of minipreps of our available cell stocks. Midipreps were unsuccessful, likely due to centrifuge limitations. LR reactions to generate more usable promoter-gene pair parts continued, expanding to include the CI434-VP16, LexA-VP16, and Mnt-VP16 genes, whose expressed proteins activate the appropriate corresponding minCMV promoters. Most of the LRs done were not successful, reason unknown at this time.

Week 3: June 20 - June 24

In the third week we continued expanding our library of usable DNA parts by creating more combinations of genes and promoters using the LR reaction.

Week 4: June 27 - July 1

In the fourth week we implemented a color-coded box system in our -20 freezer to accommodate for the growing need of organization of the growing number of DNA parts. We began learning and using the Gibson reaction to create the gene part AVPR2-TEVs-GV16, which is expressed to produce a vasopressin receptor bound to Gal4-VP16 by a TEV sequence that can be recognized by the TEV Protease. Gibson reaction results were not great, and it appeared that our AVPR2 DNA was not of sufficient quality, so we re-PCRed the AVPR2 DNA segment.

Week 5: July 4 - July 10

We began looking into using the Goldengate assembly method, but two gene elements contained a cut site that needed to be mutated out. We performed Site-Directed Mutagenesis using the Lightning Kit in the hopes of mutating out the cut site, but results were not successful due to mishandling during the protocol, so the procedure was set to be repeated. Gibson assembly of AVPR2-TEVs-GV16 continues as we re-PCRed the AVPR2 DNA segment and re-run the entire Gibson protocol, picking 20 colonies in a determined attempt to obtain a successful result.

Week 6: July 11 - July 17

In the sixth week we continued re-attemping previous failures using modified protocols or higher quality DNA in hopes of obtaining successful reactions. We also began looking at Cadherins and the possibility of using them as a clumping mechanism for mammalian cells. In order to visualize Cadherins, we needed some sort of fluorescent color, so construction of NCadherin-EGFP (NCadherin is one of many types of cadherin) began. DNA for NCad-EGFP was ordered, but needed to be in a format such that we could LR react it with the different promoters we have. This is done by attaching attB sites to flank the NCad-EGFP gene and then performing the BP reaction, which generates LR reaction-compatible parts. PCR of the attB sites was successful. By this week we have also begun work with mammalian cell cultures. To explore the limitless possibilities of synthetic biology, a few of us took it upon themselves to look into other interesting gene components, such as the Caspase gene and the FF4 tag.

Week 7: July 18 - July 24

Part of the team worked on creating protocols for a robot liquid handler to run the usual lab reactions that we run. We are hoping that the robot can replace us and do our liquid-related lab work for us. Various dry test runs were done. We transfected various DNA parts that we have into Hek293 cells, and results show that most of our DNA works. We investigated the TRE-rtTA3 system as well as the UAS-Gal4 system. Both seemed to be functional.

Week 8: July 25 - July 31

This week we underwent a momentous drive to create more LRs. A large list of promoter-gene pairs was conceived of and we began to run through LR reactions in somewhat of a factory manner. This occupied much of our time. We also received and prepared DNA parts from Elowitz's group in Caltech. We also got trained on using the FACS machine and began to get quantitative data on our transfections.

Week 9: August 1 - August 7

In the ninth week we did a lot of internal re-organization to increase our overall efficiency in work. This involved re-organizing an internal wiki that we use for management of our available DNA as well as a log of transfections needed to be done. Lots of samples were FACS-ed, generating lots of results that we can make graphs out of. Many of our parts were successfully characterized.

Week 10: August 8 - August 14

Computer simulations of the Notch-Delta interactions were presented in our group this week, and we became convinced of the possibility of creating self-patterning mammalian cells. On the DNA side of things, we are trying to create more and more DNA using miniprep, because midipreps have for some reason not been successful for us or the 2010 iGEM team. On the transfection side, a lot of new DNA parts were transfected, observed under the microscope, and FACS-ed to quantify their effect/behavior.

Week 11: August 15 - August 21

In our experimental attempt to characterize the Notch-Delta interaction, we used co-culture of CHO and Hek293 cells as well as stable cell lines of Notch-containing and Delta-containing cells from Elowitz to observe the trans-activation of Notch by Delta. Other experiments using CHO cells, which are more difficult to transfect than Hek293 using Lipofectamine, were carried out to observe the behavior of CHO transfected cells.

Week 12: August 22 - Later

Stay tuned!

Week 12

September 1

Grant: Inoculations of the aforementioned LR constructs.

August 29

Charles - CHLRs see CH notebook. H, HL, T, U and delta-ff4, H and T for ncad-2a-eyfp. inoculated propagations for HLNcad2Aeyfp, UASNcad2aeyfp, and Tre:delta.

August 28

Grant: Restarted LR reactions of 9 of the constructs listed on the Workflow (only the pEXPR_12_Hef1a-LacO_Notch-CI434-VP16 construct succeeded, possibly due to incubation time for LR reactions). Started PCRs for Tango constructs using new padding for TEV cut site.

August 25

Divya: Won. (See Personal Notebook for details).

August 23

Charles - FACS Kens 50 50 and 80 20 Hek TRE-delta senders and CHO receivers. FACS was clean. Cells were all healthy. Lowered voltage of DAPI channel from 300 to 240 to move blank cells basal fluorescence to the bottom left corner. Laser was on, everything went according to plan. Preliminary results look promising, with increasing dox for each coculture, there was increasing FITC channel signal. Preliminary results suggest that our delta works.

August 22

Charles - FACS of Kens coculture stuff. Ambiguous results.