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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.

Label	Gel	Legend
b		Column 0: HyperLadder Column 1-3: pEXPR_3-4_minCMV4xLexA_EYFP Column 4-6: pEXPR_3-4_minCMV4xMnt_EYFP Column 7-9: pEXPR_3-4_minCMV4xCI434_EYFP Column 10-12: pEXPR_3-4_minCMV4xHef1ALacO_EYFP Column 13-15: pEXPR_4-5_TRE_mKATE Column 15-18: pEXPR_1-2_Hef1A_EBFP

June 13, 2011

Status

DNA	Status	Comment
Mntcmv/1x cI434:eYFP	good
Mnt cmv/4x LexA:eYFP	bad
HefIa/LacO:eYFP	good
Tre:LacI Krab	good
HefIaL EBFP2	bad

Date	Assignee	DEST_R4R2	L4-R1 Promoter	L1-L2 Gene	LR?	Tube Number	Colony Number, Antibiotic, Person	Protocol Used
6.13.2011	Jon	2-3	Tre	C1434-VP16	Yes. 6-13, 11am	10	about 50, AMP, JC	No
6.13.2011	Tyler	2-3	Tre	LexA-VP16	Yes. 6-13, 11am	9	about 30, AMP, GR	No
6.13.2011	Mariola	2-3	Tre	Mnt1-VP16	Yes. 6-13, 11am	Eppendorf	0, AMP, GR	No
6.13.2011	Semon	4-5	Tre	mKate	Yes. 6-13, 11am	11	about 10, AMP, SR	No
6.13.2011	Divya	3-4	minCMV-7xMnt1	eYFP	Yes 6/13, 1:30pm	?	1000s, AMP, GR	No
6.13.2011	Jenny	3-4	minCMV-4xMnt1	eYFP	Yes 6/13, 1:30pm	?	1000s, AMP, GR	No
6.13.2011	Jenny	3-4	minCMV-4xC1434	eYFP	Yes 6/13,1:30pm	?	1000s, AMP, GR	No

June 14, 2011

Highlights:

~10:30 AM: Miniprep of pDEST23-ccdB commenced and completed. DNA concentrations of the two samples were 175 and 310 ng/uL

~1:00 PM: Inoculation of cultures for pEXPR-23-TRE-CI434-VP16, pEXPR-23-TRE-LexA-VP16, pEXPR-45-TRE-mKate, pEXPR-34-minCMV-7xMnt-eYFP, pEXPR-34-minCMV-4xMnt-eYFP, and pEXPR-minCMV-4xCI434 were conducted. Colony counts from the previous transformations are noted above.

~1:30 PM: LR Gateway reactions were performed for the failed pEXPR-23-TRE-Mnt-VP16 and the new pEXPR-12-Hef1A-rtTA.

~5:00 PM: Inoculation of midiprep cultures for EXPR-34-minCMV4xLexA-eYFP, two samples of pEXPR-34-Hef1ALacO-eYFP, pEXPR-12-Hef1A-EBFP, pEXPR-23-TRE-LacIKrab, and pEXPR-34-minCMV1xcI434-eYFP commenced. We plan to midiprep the cells at about 10:00 AM.

~6:00 PM: Transformation of the aforementioned LR Gateway reactions commenced. Transformation completed around 9:00 PM.

~7:00 PM: Design for the TANGO primers complete.

June 15, 2011

~4AM: Grant did more LRs – 3-4_minCMV-4xLexA:eYFP, 1-2_Hef1a:rttA3, 1-2_Hef1a:eBFP2

~10AM: Minipreps done on the 6 LRs that succeeded from yesterday by Grant

Nanodrops

Date	Assignee	Vector	Concentrations (ng/uL) *denotes 260/280 below 1.8
6.14 11AM	Jon/Michelle/Clara	2-3_Tre:C1434-VP16	A: 284.0* (1.78), B: 391.3, C: 380.9, D: 205.5
6.14 11AM	Jon/Michelle/Clara	2-3_Tre:LexA-VP16	A: 361.4, B: 193.9* (1.64), C: 212.9* (1.58), D: 390.6
6.14 11AM	Jon/Michelle/Clara	4-5_Tre:mKate	A: 176.9, B: 180.3, C: 188.7, D: 208.8
6.14 11AM	Jon/Michelle/Clara	3-4_minCMV-4xC1434:eYFP	A: 173.8, B: 152.5* (1.60), C: 319.5, D: 192.5
6.14 11AM	Jon/Michelle/Clara	3-4_minCMV-7xMnt1:eYFP	A: 147.7, B: 251.5, C: 147.6, D: 178.2
6.14 11AM	Jon/Michelle/Clara	3-4_minCMV-4xMnt1:eYFP	A: 195.1, B: 241.2* (1.75), C: 193.7, D: 157.0

Restriction Maps, 6.14 5PM

Assignee	DNA	Enzyme	Expected Results	Comments
Jon	3-4_minCMV-4xMnt1:eYFP	SalI-HF	5288bp, 2044bp if works; 5288bp, 1600bp, 977bp if doesn't	All: 1uL SalI, 2uL Buf 4, 2uL BSA; A,B,C: 3uL DNA, D: 4uL DNA; DI up to 20 uL
Jon	3-4_minCMV-7xMnt1:eYFP	SalI-HF	5288bp, 2257bp if works; 5288bp, 1600bp, 977bp if doesn't	All: 1uL SalI, 2uL Buf 4, 2uL BSA; A,B,C: 3uL DNA, D: 4uL DNA; DI up to 20 uL
Jon	4-5_Tre:mKate	SalI-HF	5288bp, 1995bp if works; 5288bp, 1600bp, 977bp if doesn't	All: 1uL SalI, 2uL Buf 4, 2uL BSA; A,B,C: 3uL DNA, D: 4uL DNA; DI up to 20 uL
Mariola	2-3_Tre:C1434-VP16	SalI-HF	5288, 1862, 412 bp if works; 5288bp, 1600bp, 977bp if doesn't	All: 1uL SalI, 2uL Buf 4, 2uL BSA; A: 1.8uL DNA, B,C: 1.3uL DNA, D: 2.4uL DNA; DI up to 20 uL
Mariola	3-4_minCMV-4xC1434:eYFP	BamHI	3757, 1855, 1166, 478 bp if works; no cut if doesn't	All: 1uL BamHI, 2uL Buf 4, 2uL BSA; A 2.9uL DNA, B: 3.2uL DNA, C: 1.6uL DNA, D: 2.6uL DNA; DI up to 20 uL
Tyler	2-3_Tre:LexA-VP16	ScaI	2 cuts: 4464 and 2729 bp if works; 3 cuts: 3535, 2729, 1589 if doesn't	All: 1uL SalI, 2uL Buf 4, 2uL BSA; A: 3uL DNA, B,D: 1.5uL DNA, C: 2.5uL DNA; DI up to 20 uL From Gel Below: A and C show the entry vector, B failed as well, D seems to have worked. There is uncut DNA around 7000 bp, but the other bands seem correct. D will be sent for sequencing.

Midipreps were done by Kenneth and Tyler for sequenced verified constructs, 2-3_Tre:LacI-Krab, 3-4_minCMV 1xCI434:eYFP, and 3-4_Hef1ALac0:eYFP. Unfortunately DNA yields were low, as shown in the table below, so minipreps will be performed tomorrow, June 16, 2011.

Date	Assignee	Vector	DNA Concentration (ng/uL)
6.15.2011	Tyler	2-3_Tre:LacI-Krab	54
6.15.2011	Kenneth	3-4_minCMV 1xCI434:eYFP	216
6.15.2011	Kenneth/Tyler	3-4_Hef1ALac0:eYFP	failed

June 16, 2011

~10am Minipreps

Date	Assignee	Vector	DNA Concentration (ng/uL)
6.16.2011	Tyler	2-3_Tre:LacI-Krab	509.1
6.16.2011	Kenneth	3-4_minCMV 1xCI434:eYFP	89.1
6.16.2011	Kenneth/Tyler	3-4_Hef1ALac0:eYFP	385

June 17-18, 2011 Construction

Protocol for all Cells: Added DNA 5 minutes after removing cells from freezer. Incubated for 30 minutes. Heat shocked for 30 seconds at 42 Celsius. Incubated on ice for two minutes. Added 1 mL of SOC. Grew at 30 C for 2 hours. Plated 100 uL from total reaction on one half of plate & the rest of the reaction on the other half of plate.

Date	Assignee	DEST_R4R2	Colonies (low/high conc.)
6.18.2011	Grant	pEXPR12-Hef1A-eBFP2	Order of <10
6.18.2011	Grant	pEXPR12-Hef1A-rtTA3	Order of <10
6.18.2011	Grant	pEXPR23-TRE-CI434.VP16	Order of 100-1000
6.18.2011	Grant	pEXPR23-TRE-LexA.VP16	Order of 100-1000
6.18.2011	Grant	pEXPR23-TRE-Mnt.VP16	Order of 100-1000
6.18.2011	Grant	pEXPR34-minCMV.4xCI434-EYFP	Order of 1000-10000
6.18.2011	Grant	pEXPR34-minCMV.4xLexA-EYFP	Order of 1000-10000
6.18.2011	Grant	pEXPR34-minCMV.4xMnt-EYFP	Order of 1000-10000
6.18.2011	Grant	pEXPR34-minCMV.7xMnt-EYFP	Order of 1000-10000
6.18.2011	Grant	pEXPR45-TRE-mKate	Order of <10

Conclusion: pDEST12 appears to be growing slowly in all cases, as does pDEST45. pDEST23 grows at the rate typically expected by Gateway reaction standards while pDEST34 grows at a rate much faster than these standards. Corroborated in Weiss lab?

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.

Team:MIT/Notebook/