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.
(Lucija) 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.
(Fedja) Cloning repressor reporters 7xNicTAL+7xTAL95 BS-CMV-mCit.

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

(Lucija, Anja) 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.
(Uroš) :-( Performing multiple PCR-ligations trying to join TAL molecule and VP16 activation domain.
(Fedja) Cloning repressor reporters 7xNicTAL+7xTAL95 BS-CMV-mCit.

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

(Anja, Lucija, Uroš) Cloning of TAL regulators.
(Fedja) Unsuccessful cloning of activating reporters 7xNicTAL+7xTAL95 BS-pMin-mCit.

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

(Anja, Lucija, Uroš) Performing multiple colony-PCRs.
Repeatedly trying to get less back ligations.
(Fedja) 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"

(Uroš) First samples of TAL regulators were send for sequencing.

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

(Uroš, Anja, Lucija) First TAL activators were cloned and their sequence confirmed.
Second round of TAL regulators send for sequencing.
(Zala) Performing PCR of TAL-A, TAL-B, TAL-C, TAL-D, TAL-A:KRAB, TAL-B:KRAB, TAL-C:KRAB, TAL-D:KRAB and T-sapphire.
(Boštjan) 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"

(Anja) 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.
(Uroš)Stopped with colony PCR, start performing more straightforward method of cloning.
Multiple isolation (isolating around 20 clones for one construct and performing control restriction).
(Zala) Start cloning (Gibson Assembly) 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.
(Boštjan) 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"

(Uroš) More samples of Tal regulators sent for sequencing.
(Lucija) Cloning KRAB in front of TALs using PCR ligation.
(Anja) 3 Point ligation and Gibson Assembly.
(Boštjan) 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"

(Anja, Lucija, Uroš) Sequence analyzing.
Getting angry and depressed.
(Boštjan) 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"
(Urban) 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.
(Fedja) 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.
(Boštjan) 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"

(Urban) 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.
(Zala) Successfully cloned TAL-A:KRAB, TAL-D:KRAB, TAL-B:KRAB with T-Sapphire in pcDNA3 vector. Send for sequencing. Start cloning mNeptune.
(Boštjan) 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"

(Urban) 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.
(Anja) KRAB was cloned in front of TAL-B:KRAB, TAL-C:KRAB, TAL-B, TAL-C, and TAL-D.
(Boštjan) 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!
(Zala) Start cloning TAL-N and TAL-N:KRAB with T-Sapphire in pcDNA3 vector. Send for sequencing. PCR for new construct: TAL-A:KRAB, TAL-B:KRAB, TAL-C:KRAB, TAL-D:KRAB and mCitrin and mNeptune.

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

(Lucija) Testing repression (in HEK293 cells) using KRAB:TAL, TAL:KRAB and KRAB:TAL:KRAB constructs.
(Miha) 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
(Boštjan) 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

(Zala) Start cloning of mNeptun-mNeptun-NLS and mCit-mCit-NLS (3 point ligation) in pcDNA3 vector with different binding sides and 10xAC-pCMV-TAL-B:KRAB-t2A-mNeptune and 10xCB-pCMV-TAL-A:KRAB-t2A-mCit (unsuccessfully).

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

(Anja) Cloning of mGMK_TK30 into the pcDNA3 vector.
(Lucija, Miha) 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.
(Zala) New YFP: SEAP isolation, PCR. Again trying to clone 10xAC-pCMV-TAL-B:KRAB-t2A-mNeptune and 10xCB-pCMV-TAL-A:KRAB-t2A-mCit.
(Boštjan) 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"

(Lucija) Testing repression (in HEK293 cells) using KRAB:TAL, TAL:KRAB and KRAB:TAL:KRAB constructs with different TALs.
(Anja) 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.
(Lucija, Miha) Testing of the Pristinamycin and Rapamycin induction systems, using fluorescent protein (Citrine and Cerulean) and SEAP (secreted alkaline phosphatase) reporters.
(Uroš) Testing activation of minimal promoter with TAL:VP16 activators using fluorescent proteins.
(Boštjan, Zala) 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.

As our computer scientists were making their first steps at the lab, (Martin) was already running around with an opened Erlenmeyer flask that just came out of the autoclave. "I thought it would cool down more quickly... "

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

(Martin, Dušan) Deterministic models and analysis were completed.
(Anja, Lucija) Testing of the CMV-mGMK_TK30/GCV system on HEK293 cell with confocal microscope and flow cytometry.
(Miha) 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.
(Urban) 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.
(Zala) 3 point ligation for YFP-YFP-NLS constructs did not work – new strategy – V5 vector (Blue script). New primers for KOZAK.
(Boštjan) 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

(Urban) Sequencing confirmed that alginate lyase was assembled and cloned properly into pSB1C3 vector. :-)
(Anja) 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.
(Urban, Lucija, Miha) 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.
(Zala) Cloning in Blue script vectors: KOZAK-mCit-mCit-NLS and KOZAK-mNep-mNep-NLS.
(Boštjan) 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"

(Urban, Miha, Lucija) Testing the pristinamycine and erythromycine induction sistems with different ratios of plasmids, using SEAP reporters.
Testing Ecdyson and Rapamycin systems.
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.
(Urban) Cloning alginate lyase into vector with TAL-A and TAL-B binding sites (10x[AB]_pCMV_Aly) and expression in HEK293T.
(Zala) Incorporation of KOZAK-YFP-YFP-NLS in different vectors: 2xDN; 4xDN, 7xDN, 12xD, 12XN, 10xAC, 10xAB, 10xCB.
(Boštjan) 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.
(Everyone) Preparing the questionnaire about the use of new therapeutic systems for biopharmaceuticals for patients with hepatitis C.

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

(Anja) Testing CMV-mGMK_TK30/GCV system on HEK293 cell with optical microscope and flow cytometry.
(Lucija, Miha) 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.
(Urban) 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.
(Dušan) Stochastic models and analysis were completed.
(Boštjan) 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.
(Zala) Successfully incorporated KOZAK-YFP-YFP-NLS in vectors with different binding sites, send for sequencing. Starting also to clone pGL4.16 vector, TAL-A:VP16, TAL-B:VP16, TAL-B:KRAB – t2A – Nep, TAL-A:KRAB – t2A – mCit (PCR, gel electrophoresis).

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

(Anja) 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.
(Miha) 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.
(Urban) 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. (Boštjan, Zala) We give up trying to construct plasmids for + feedback switch that have TAL:KRAB as well as TAL:VP16. Instead we decide to separate it on their own plasmids, however maintaining same binding sites. Our + feedback switch will consist of following plasmids : 10xAC-pMin-TAL-B:KRAB, 10xAC-pMin-TAL-A:VP16-t2a-tagBFP , 10xCB-pMin-TAL-A:KRAB-t2a-mCitrine and 10xCB-pMin-TAL-B:VP16 . We succeed the first time :)

5 to go
27.8. – 2.9. "ELUDING EFFECTORS"

(Lucija) Cloning efector IFN-a into pSB1C3 vector and Gibson Assembly of VEGF_p2a_PDGF and pSB1C3 vector.
(Anja) Testing citotoxity of NK-92 cells on HEK293T expressing MICA and GFP with flow cytometry.
(Lucija, Miha, Uroš) Mutual repression switch was tested using flow cytometry and confocal microscope.
Rapamycin and Ecdyson systems were tested in pgl4 vectors, with Citrine and Cerulean reporters.
(Urban) Successfully isolated genomic DNA from the terrifying P. aeruginosa PAO1 (biosafety level 2).
Like last week, the encapsulated cells were stained to estimate their viability.
Mature P. elyakovii alginate lyase was PCR-amplified without bacterial secretion signal sequece. (Boštjan) While the tests of the plasmids for + feedback switch are performed in HEK-T cells, binding sites for ZFHD (rapalogue inducible system) are also cloned instead of 10xAC binding sites. We also try to clone EGSH (ecdysone inducible system) instead of 10xCB , but fail. Gibson assembly is again letting us down.
(Maja) Final pharmacokinetic model for hepatitis.

4 weeks (1 month) to go
3.9. – 9.9. "KILLER CELLS have licence to KILL"

(Lucija) Testing TAL activators and repressors using different reporters.
(Anja) Testing citotoxity of NK-92 cells on HEK293T expressing MICA and BFP with flow cytometry.
(Lucija, Miha) Testing Pristinamycin and Tetracyclin systems in pcDNA3, with fluorescent reporters and double luciferase assay.
Testing the Rapamycin system in pgl4, with fluorescent reporters.
First prove the switch based on positive feedback loop should work.
Fluorescence was measured with the 96 well fluorescence plate reader.
(Urban) Primers for full-length P. elyakovii and P. aeruginosa alginate lyase without bacterial signal sequence arrived.
Full-length P. elyakovii and P. aeruginosa along with mature P. elyakovii alginate lyase were cloned downstream of constitutive promoter.
The encapsulated cells were stained to estimate their viability.
Recombinant alginate lyase was added to alginate-PLL microcapsules in enormous concentrations: nothing happened. :O
Starting cloning of our constructs into the biobrick system.
(Boštjan) After advice of our Head Advisor (The Boss) we try to clone mCit downstream of TAL-B:VP16, instead of TAL-A:KRAB. The strategy is the same as the one fortnight ago. This time, however, we don't succed. We get a bunch of recombinant clones, and have no idea what is happening.

(Zala, Fedja) Starting to distribute questionnaires to the patients.

3 to go
10.9. – 16.9. "NEW and MORE MODELING and PHOTOsession"

(Dušan) C#Sim algorithm and models were completed. Real-time 3D visualization of pharmacokinetic model more or less completed.
PHOTO of the team.
(Anja) Testing citotoxity of NK-92 cells on HEK293T expressing MICA and BFP with flow cytometry.
(Miha) Testing the Erythromycin system in pcDNA3, with fluorescent reporters.
(Lucija, Miha, Uroš) Positive feedback loop switch was tested with flow cytometry and confocal microscope.
(Urban) Cells were transfected with whole alginate lyase arsenal and their supernatants/lysates were blotted. We are still having problems with protein production. :(/
The encapsulated cells were of course stained again to estimate their viability. They're just not interested in dying.
(Boštjan) Started cloning "effectors" - genes for therapeutic proteins anakinra, interferon alpha, hepatocyte growth factor, pdgf and vegf. We aim to clone it into plasmids for + feedback switch instead of fluorescent reporters. Using Gibson assembly we fail, which we ought to expect, considering all of the previous failures with it. Also tried to clone mCit downstream of VP16 as mentioned using two cycles of restriction and ligation (aka ReLi :). First cycle succeeds, the second one unfortunately not.

2 to go
17.9. – 23.9. "WIKI rush and movie making and BIOBRICK deposit"

Last encapsulation experiment. Alongside alginate-PLL microcapsules, we also produced alginate beads with immobilized cells.
Testing the switch.
(Urban) Real-time recording of alginate bead degradation with alginate lyase. We did not expect that based on what we have seen so far. There is no easy way to get to the result you sought all this time ... you are very close, though. For the last time, the first batch of encapsulated cells was dyed again to observe its viability. Cells are very much alive and kicking.
Effector testing.
(Fedja) BioBRICK action.
(Boštjan) Tried to clone effectors into + feedback switch again, but this time using ReLi (restriction and ligation). We again get a bunch of nonproper clones. We are guessing that the pGL vector we are using might be responsible for it.

1 to go
24.9. – 30.9. "WIKI FREEZE"

(Dušan) Copy-pasting this text to this public wiki at 3:00 a.m. Coping with tunnel vision.

The week!!
1.10. – 7.10. "The week - Amsterdam we are coming"



3 to go
8. 10. - 14. 10. "Mission: Switch it!"

(Uroš, Urban, Miha, Anja, Lucija) As soon as we got off the plane from Amsterdam, we started planning for experiments to further characterise our switch - especially to switch it!
The experiments were preformed using HEK293T cells on flow cytometer, confocal microscope and luciferase and SEAP assays.
(Zala) Start cloning 10xAC-pMin-SEAP and 10xCB-pMin-SEAP.
(Martin) Incorporating experimental measurements of TAL regulators into the models turned out to be useful. Resulting predictions were surprisingly very realistic.< br/>

2 to go
15. 10. - 21. 10. "Can we make it?"

(Uroš, Urban, Miha, Anja, Lucija) Switching experiments are under way an new ones are constantly being designed. We are patiently awaiting THE RESULT.
(Urban) Alginate beads were prepared for alginate lyases' activity assays.
HEK293T were transfected to produce our whole arsenal of alginate lyases.
Alginate lyase-containing growth media were concentrated and alginate beads were incubated in these concentrated cell supernatants over weekend.
(Martin, Dušan) Simulating different activator-repressor ratios.

1 to go
22. 10. - 28. 10. "Mission: accomplished and WIKI FREEZE - again?!"

(Uroš, Urban, Miha, Anja, Lucija) Last set of experiments and assays with our genetic switch.
(Urban) Confocal microscopy of half-digested alginate beads.
(Zala) Successfully cloned plasmids 10xAC-pMin-SEAP and 10xCB-pMin-SEAP in pGL4.16.

Next: Lab safety >>