Revision as of 00:13, 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: