Revision as of 00:38, 25 September 2012 by Dusanv (Talk | contribs)


Before 11.4. "in silico EXPERIMENTS"
Discussing what plasmids, primers, enzymes, and chemicals we need.

25 weeks to go
11.4. "start the WET LAB work"

We obtained TAL-A, TAL-B, TAL-C, TAL-D, and Nic-TAL from the AddGene and from Registry and KRAB and VP16 from the lab sources.
Plasmids were transformed into DH5a and the bacteria were plated on ampicilin plates.
Single colonies were inoculated into minipreps.
We isolated the plasmids in larger quantities.
Competent cells (DHA5α, DB3.1) were prepared.
Preparation of LB media.
Cloning of TAL regulators (TAL+VP16).
Primers ordering.
Cloning repressor reporters 7xNicTAL+7xTAL95 BS-CMV-mCit.

24 weeks (6 months - half a year) to go
16.4. - 22.4. "TAL week"

TAL-A, TAL-B, TAL-C, TAL-D, Nic-TAL and KRAB fragments were amplified using PCR.
We tried to ligate KRAB upstream, downsteram and on both positions of all TAL constructs.
PCR ligation was used, but failed repeatedly.
:-( Performing multiple PCR-ligations trying to join TAL molecule and VP16 activation domain.
Cloning repressor reporters 7xNicTAL+7xTAL95 BS-CMV-mCit.

23 to go
23.4. - 29.4. "another TAL week"

Cloning of TAL regulators.
Unsuccessful cloning of activating reporters 7xNicTAL+7xTAL95 BS-pMin-mCit.

22 to go
30.4. - 6.5. "and another TAL week"

Performing multiple colony-PCRs.
Repeatedly trying to get less back ligations.
Another week of unsuccessful cloning of activating reporters 7xNicTAL+7xTAL95 BS-pMin-mCit.

21 to go
7.5. - 13.5. "light at the end of tunnel"

First samples of TAL regulators were send for sequencing.

20 weeks (5 months) to go
14.5. - 20.5. "TAL success-I"

First TAL activators were cloned and their sequence confirmed.
Second round of TAL regulators send for sequencing.
Went straight to performing PCR for cloning mCit into pGL vector. A note from one of our advisors:

19 to go
21.5. - 27.5. "Gibson ASSEMBLY-the revelation"

KRAB was ligated in front of TAL-A:KRAB using Gibson Assembly.
We cloned TAL-A:KRAB, TAL-B:KRAB, TAL-C:KRAB downstream from minimal promoter.
Stopped with colony PCR, start performing more straightforward method of cloning.
Multiple isolation (isolating around 20 clones for one construct and performing control restriction).
Cloning of TAL-A, TAL-B, TAL-C, TAL-D and TAL-A:KRAB, TAL-B:KRAB, TAL-C:KRAB, TAL-D:KRAB ligated to T-sapphire in pcDNA3 vectors.
It turned out mCit in pGL vector was not okay. With mentors we designed another strategy, so we PCRed and ligated again.

18 to go
28.5. - 3.6. "three point LIGATIONS"

More samples of Tal regulators sent for sequencing.
Cloning KRAB in front of TALs using PCR ligation.
3 Point ligation and Gibson Assembly.
Ligated different TAL:KRABs into pcDNA3 vector with CMV promoter and binding sites for TAL effector. We call these 2nd level plasmids-they represent 2nd level of logic and we annotate them in a form of »7xTAL_A-pCMV-TAL_A:KRAB« . At this time our plasmids have only 7 repetitions of TAL binding site upstream of promoter.

17 to go
4.6. - 10.6. "another TAL week"

Sequence analyzing.
Getting angry and depressed.
Together with Rok (The Cloning Master – by Tina J) we designed primers for PCR amplifying 6 repetitions of TAL binding sites = 6x TALBS, which we will ligate together to get 12xTALBS. We succeed the first time.

16 weeks (4 months) to go
11.6 - 17.6. "LUCIFERASE-glowing in the dark"

We started working with mammalian cell cultures (HEK293).
First experiment to take place was double luciferase assay to quantify activation of firefly's luciferase expression under minimal promoter using TAL-D:VP16.
Testing activation of minimal promoter with TAL:VP16 activators using fluorescent proteins.
Cloning of new NicTAL and NicTAL-KRAB. Not successful.
We got synthetic genes for binding sites for TAL_A, TAL_B and TAL_C combined to form 7 doubles, in each double there is one BS for TAL_A and one for TAL_B and designated it as 7x(AB) . We get three combinations : 7x(AB), 7x(AC), 7x(CB). We ligated this sequence into pCMV-mCit plasmid and we got 7x(AB)-pCMV-mCit, for example.
Previous week we got 12xBS sequence ligated from two 6xBS sequences. This week we ligated them into pCDNA3, but restriction digest show no appropriate bands, just smears:

15 to go
18.6. - 24.6. "New actor in a play-ALGINATE LYASE"

We continued testing activation achieved with TAL-D:VP16 using HEK293 cells.
Last week's relative luciferase units (RLUs) were low, so we increased the amount of reporter plasmid.
We selected alginate lyase (Aly) from Pseudoalteromonas elyakovii as the most appropriate enzyme for degradation of microcapsules and ordered a synthetic codon-optimized version of this gene.
Preprotrypsin leader sequence was also added to the 5' end and Myc-tag was added to 3' end of Aly.
The whole sequence was broken down into three gBlocks which could be put togheter using Gibson assembly.
Really hope this will work as good as it did for Cambridge back in 2010.
We got primers for amplifying 5 repetitions of binding sites for TAL A, B and C, with pretty much the same strategy we ligated them together to get 10x(two TAL BS), eg. 10x(AB) or 10x(AC) or 10x(CB).We also ligated apropriate TAL:KRABs into pcDNA3 plasmids with different binding sites with 7 repetitions:

  • (AB)x7-pCMV-mCit
  • (AC)x7-pCMV-mCit
  • (AB)x7-pCMV-luc
  • (AB)x7-pCMV-TAL_C:K
  • (AB)x7-pCMV-TAL_D:K
  • (AC)x7-pCMV-luc
  • (AC)x7-pCMV-TAL_A:K
  • (AC)x7-pCMV-TAL_C:K
  • (AC)x7-pCMV-TAL_D:K
  • (AC)x7-pCMV-TAL_B:K
Note: K means KRAB

14 to go
25.6. - 1.7. "VARIATION in TAL repressors"

We started testing NOR gates using double luciferase assay in HEK293 cells.
Constructs used were TAL-A:KRAB and TAL-C:KRAB.
Reporter plasmid had binding sites for both TAL regulators used upstream of fLuc under control of CMV promoter.
KRAB was cloned in front of TAL-B:KRAB, TAL-C:KRAB, TAL-B, TAL-C, and TAL-D.
By the end of this week we also successfully constructed 10xAC-pCMV-mCit, 10xAB-pCMV-mCit and 10xCB-pCMV-mCit reporter plasmids.
We once again tried to construct 12xNicTAL and 12xTAL_D binding sites in the pcDNA3 vector via 3 point ligation. Out of ten isolated clones for each construct we got 1 correct after restriction digest. Success!

13 to go
2.7. - 8.7. "Pristinamycin from FRANCE"

Testing repression (in HEK293 cells) using KRAB:TAL, TAL:KRAB and KRAB:TAL:KRAB constructs.
Cloning of TAL-B:KRAB into the pMF208 vector, downstream of the pSV40-PIR promotor to place it under control of the Pristinamycin inducible system with restriction and ligation method.
Cloning of TAL-A:KRAB into the pZFHD1-1 vector to place it under control of the Rapamycin inducible system, using restriction and ligation.
Cloning of two fluorescent reporters
Successfully cloned 2nd level construct with 10x Binding sites, as well as firefly luciferase reporter plasmids with these binding sites. These are still good days regarding cloning.

  • (AB)x10-pCMV-TAL_D:K
  • (AB)x10-pCMV-fLuc
  • (AC)x10-pCMV-TAL_D:K
  • (AC)x10-pCMV-fLuc
  • (CB)x10-pCMV-TAL_D:K
  • (CB)x10-pCMV-fLuc
  • (AC)x10-pCMV-TAL_B:K
  • (CB)x10-pCMV-TAL_A:K

12 weeks (3 months) to go
9.7. - 15.7. "Another happy couple TYMIDIN KINASE and GUANYLATE KINASE - virus and mouse"

Cloning of mGMK_TK30 into the pcDNA3 vector.
First tests of the Pristinamycin and Rapamycin inducible sistems.
E was amplified from E-VP16 using PCR and ligated into pMF207 to create E:KRAB, for the erythromycin inducible system.
Starting of cloning 2-fluorescent proteins (Neptune-Neptune, Citrine-Citrine) with NLS in pcDNA3 vectors with different binding sides.
Started performing Gibson assembly to construct above plasmids with fluorescent reporters linked via t2a sequence. One of the plans:

11 to go
16.7. - 22.7. "COLORFUL science-citrine, cerulean"

Testing repression (in HEK293 cells) using KRAB:TAL, TAL:KRAB and KRAB:TAL:KRAB constructs with different TALs.
Monitoring of HEK293 cells transfected with CMV-mGMK_TK30 and incubated with gancyclovir for different time periods by optical microscope.
SEAP was cloned into reporter vectors with different binding sites for TALs.
Testing of the Pristinamycin and Rapamycin induction systems, using fluorescent protein (Citrine and Cerulean) and SEAP (secreted alkaline phosphatase) reporters.
Testing activation of minimal promoter with TAL:VP16 activators using fluorescent proteins.
Still performing Gibson assembly to construct pCMV-TAL-A:K-mCit and pCMV-TAL-B:K-mNeptune reporter/represor plasmids - we need these for mutual represor switch.

10 to go
23.7. - 29.7. "MODELLING week"

Deterministic models and analysis were completed.
Testing of the CMV-mGMK_TK30/GCV system on HEK293 cell with confocal microscope and flow cytometry.
Measuring SEAP activity in supernatants of cells transfected with vectors with TAL binding sites.
Testing of the Pristinamycin and Erythromycin induction systems, using fluorescent reporters.
Gibson assembly of synthetic alginate lyase from Pseudoalteromonas elyakovii and ligation in pSB1C3 vector.
Cloning TAL-A, TAL-B, TAL-C with fast degradation tags.
After numerous Gibson assemblies and many colony PCRs we finally have plasmids for mutual repressor switch. Planning construction of positive feedback switch plasmids:

9 to go

Sequencing confirmed that alginate lyase was properly assembled.
Testing of the mGMK_TK30/GCV system on HEK293 cell with optical microscope and flow cytometry.
Cloning of TAL-A:KRAB, TAL-B:KRAB and Nic-TAL:KRAB into the pEGSH vector to place it under control of the ecdysone inducible system, using restriction and ligation.
Testing of the Pristinamycin, Rapamycin and Erythromycin induction systems, using double luciferase assay, fluorescent proteins and SEAP.
Cloning of BFP in reporter plasmids and using Gibson assembly to create TAL effectors and BFP linked with t2A peptide.
Sequencing confirmed that alginate lyase was assembled and cloned properly into pSB1C3 vector. :-)
Performing Gibson assembly for positive feedback switch, we are assembling a pGL vector, TAL:KRAB, TAL:VP16 and fluorescent reporter linked via t2a in one reaction. We repeatedly get no colonies on LBA plates.

8 weeks (2 months) to go
6.8. - 12.8. "New colors - Neptun and BFP makes science pretty"

Testing the pristinamycine and erythromycine induction sistems with different ratios of plasmids, using SEAP reporters.
Amplifying tTR and TRE with PCR and ligating into pMF207 and pMF208 respectively to prepare tTR:KRAB and pSV40-TRE for the Tetracycline system.
Testing of BFP, mNeptune and mCitrine with flow cytometry.
First tests of the mutual repressor switch with Citrine/Neptune and Cerulean reporters or double luciferase assay.
Testing Ecdyson and Rapamycin systems.
Cloning alginate lyase into vector with TAL-A and TAL-B binding sites (10x[AB]_pCMV_Aly) and expression in HEK293T.
We rearrange the parts in Gibson cloning strategy for + feedback switch. We start to get colonies, some even seem okay after colony PCR, but not after restriction digest.

7 to go
13.8 - 19.8. "SWITCH to test - not electric"

Testing CMV-mGMK_TK30/GCV system on HEK293 cell with optical microscope and flow cytometry.
Testing mutual repressor switch and Ecdyson system, with Citrine/Neptune and Cerulean reporters or double luciferase assay.
First experiments with TAL effectors that mutually repress each other and activates itself on separate plasmids.
Western blot of alginate lyase-expressing cells' supernatant and detection with anti-Myc antibodies.
Seems like there's not much of alginate lyase in supernatant, probably because there is still bacterial signal sequence downstream of eucaryotic.
We will order primers that will contain only preprotrypsin leader sequence.
Stochastic models and analysis were completed.
We still don't get proper constructs for + feedback loop switch. Several strategies of Gibson assembly are performed by 5 students. As well as changing strategies we try varying reaction conditions and using different strains of competent cells.
Incorporation of KOZAK-YFP-YFP-NLS in different vectors with binding sides: 2xDN; 4xDN, 7xDN, 12xD, 12XN, 10xAC, 10xAB, 10xCB.

6 to go
20.8. – 26.8. "A LOT of NEW STAFF"

Testing CMV-mGMK_TK30/GCV system on HEK293 cell with optical and confocal microscope.
Cloning MICA into pcDNA3 vector.
Cloning CMV-mGMK_TK30 into pSB1C3 vector.
Cloning of constructs for bistable switch with positive feedback loop in minimal promoter vectors.
Testing Tetracycline inducible system, using SEAP reporter.
Mutual repression switch was tested with flow cytometry and confocal microscope.
PIR and TRE binding sites were cloned into pcDNA3 vectors in front of TAL regulators to create Pristinamycin and Tetracyclin systems with CMV promoters.
Preparation of buffers for first encapsulation experiment.
HEK293T to be encapsulated were transfected with alginate liase and empty vector for control experiment.
First encapsulation (cells expressing lyase) was successful, while second (control) produced way too big capsules that were very unstable and would burst while pippeting.
Encapsulated cells were dyed with Hoechst and 7-AAD dye to estimate their viability.

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