iGEM 2016 — Team EPFL

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# Team:EPFL/Notebook

iGEM EPFL 2016

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## Laboratory Notebook

---

##### Week 1 (07/18/2016 – 07/24/2016)

* We received the six plasmids in bacterial stabs from Addgene.org: p404TDH3, pJZC638, PTEF1-yEGPCLN2PEST-pRS406, pCYC1m\_yeGFP, POT2-RFP, pFA6a-kanMX6-PGAL1-GFP.
* Bacteria delivered from Addgene.org in stabs were plated on selective medium with Amp; overnight culture of single colonies.
* Glycerol stocks of plasmids (all of them) received from Addgene were made first (25% glycerol).
* [Miniprep](https://2016.igem.org/Team%3AEPFL/Protocols#plasmidpurification)  of cultures with the plasmids(all of them).
* We received from IDT the eight inserts (gBlocks); gBlocks were resuspended in TE buffer.
* [PCR](https://2016.igem.org/Team%3AEPFL/Protocols#pcr)  of parental plasmids and gBlocks with the appropriate primers.

##### Week 2 (07/25/2016 – 07/31/2016)

* Restriction Analysis of 3 different plasmid to check some features.
* PCR of parental plasmids and gBlocks with the appropriate primers testing different programs.
* [PCR purification](https://2016.igem.org/Team%3AEPFL/Protocols#pcrpurification)  of the whole PCR products and quantification with Nanodrop®.
* First try of [Gibson assembly](https://2016.igem.org/Team%3AEPFL/Protocols#gibsonassembly)  for 8 of 9 plasmids needed.

##### Week 3 (08/01/2016 – 08/07/2016)

* 1L of [LB agar medium (50 plates) with ampicillin](https://2016.igem.org/Team%3AEPFL/Protocols#lbagarplate) .
* PCR and PCR purification of 2xPP7 and p404 (didn’t work the first time).
* Gibson Assembly of PCP\_Mxi1 and p404\_2xPP7, trying different protocols and different setups.
* New PCR of gblocks to try to obtain higher concentration.
* Electrophoresis gel to check plasmid and insert sizes.

##### Week 4 (08/08/2016 – 08/14/2016)

* PCR purification and quantification (using Nanodrop®) of the PCRs from previous week.
* Gibson assembly and [transformation](https://2016.igem.org/Team%3AEPFL/Protocols#transformationintocompetentcells)  of TDH3+RFP and pGal1+C6\_TDH3\_3.
* [PCR colony](https://2016.igem.org/Team%3AEPFL/Protocols#pcrcolony)  of 12 colonies for TDH3+RFP ,4 colonies for pGal1+C6\_TDH3\_3, 2 colonies for p404\_2xPP7 and one for PCP\_Mxi1.
* Inoculation of TDH3+RFP and pGal1\_GFP+c6\_TDH3\_3 (colonies that showed correct size on the gel)
* Miniprep of these colonies (previou point).
* Electrophoresis gel to check plasmid and insert sizes.
* Inoculation and miniprep of TDH3-RFP and GAL1\_GFP\_C6TDH3\_3.
* Enzymatic analysis of products: Gal1\_GFP\_c6 and TDH3\_RFP.
* Gibson assembly and transformation of PJZC638\_A + ADH1 ,pJZC638\_R + PCP\_Mxi1, Gal1+C6\_TDH3\_1 and Gal1+C6\_TDH3\_2.
* Sequencing of RFP\_TDH3, Gal1-GFP\_c6\_TDH3\_1, Gal1-GFP\_c6\_TDH3\_2 and Gal1-GFP\_c6\_TDH3\_3 at Microsynth.

##### Week 5 (08/15/2016 – 08/21/2016)

* Glycerol stocks of confirmed products (plasmids without mutation in the insert): RFP\_TDH3, Gal1-GFP\_c6\_TDH3\_1, Gal1-GFP\_c6\_TDH3\_2 and Gal1-GFP\_c6\_TDH3\_3 (25% glycerol).
* Restriction digestion TDH3-RFP and GAL1-GFP-C6-TDH3-1, 2, 3 (check the size of the whole plasmid). This is also needed for the integration in yeats as the plasmid must be linearized).
* Gibson Assembly, transformation and PCR colony on pJZC638\_A+ADH1.
* Plates test with different combinations of selections.
* Preparation of 12 ml of cultures for each confirmed product (a lot of dna needed for integration in yeasts) followed by Minipreps and quantifications with Nanodrop®.
* Gibson Assembly, transformation and PCR colonies of pJZC638\_R+PCP\_Mxi1 and p404+1xPP7.
* Inoculation of pJZC638\_R+PCP\_Mxi1 and p404+1xPP7.
* PCR followed by a PCR purification of 2xPP7.
* Gibson Assembly and transformation of p404+2xPP7 and Cas9VPR+Tef1.

##### Week 6 (08/22/2016 – 08/28/2016)

* Miniprep of pJZC638\_R+PCP\_Mxi1 and p404+1xPP7.
* Preparation of 12 ml bacterial culture in [LB medium](https://2016.igem.org/Team%3AEPFL/Protocols#lbmedium)  (with ampicillin) for pJZC638\_R+PCP\_Mxi1, p404+1xPP7 and PJZC638\_A+ADH1. (for integration in yeasts).
* Preparation of 1L of YPDA.
* PCR colony and miniprep of p404\_2xPP7.
* PCR of tef1+Trp with new primers.
* Preparation of 12 ml of bacterial culture in LB medium (with ampicillin) for pJZC638\_R+PCP\_Mxi1, p404+1xPP7, PJZC638\_A+ADH1 and CYC1\_GFP.
* [Linearization](https://2016.igem.org/Team%3AEPFL/Protocols#restrictionenzymes)  of TEF1\_GFP (repression) [integration](https://2016.igem.org/Team%3AEPFL/Protocols/#yeastsintegration) in Yeasts.
* PCR purification of TPGI and tef1.
* Gibson Assembly, transformation, PCR colony and inoculation of scRNA\_Trp+tef1.
* [Replate](https://2016.igem.org/Team%3AEPFL/Protocols#singlecolonystreak) all of the confirmed plasmids and glycerol stocks.
* Sequencing of other sequences (promoters) of pJZC638\_R+PCP\_Mxi1, p404+1xPP7 and PJZC638\_A+ADH1
* Linearization of ADH1, TDH3\_RFP and Tef1\_GFP (repression).
* Integration of TDH3\_RFP and PJZC638\_A+ADH1 in yeasts.
* Miniprep and sequencing of scRNA\_Trp+tef1, p404+2xPP7 and PCP\_Mxi1 at Microsynth.
* Inoculation, miniprep, linearization and integration of tef1\_GFP.
* Preparation of 16 ml bacterial culture with LB medium (with ampicillin) of TDH3\_RFP, Tef1-GFP and PJZC638\_A+ADH1 for integration in yeasts.

##### Week 7 (08/29/2016 – 09/4/2016)

* Pre-cultures of yeasts with RFP.
* Preparation of 50 SD plates for yeast (including 12 with Geneticin).
* Miniprep of Tef1-GFP, PJZC638\_A+ADH1, 2xPP7 and Tef1\_c3#0.
* Linearisation of Tef1-GFP and Cyc1-GFP (double digest as a positive control).
* Integration yeast with cyc\_GFP and Tef1\_GFP.
* Glycerol stock and replating of tef1\_scRNA\_Trp + 2xpp7.
* Linearization of C6\_TDH3\_1,2,3 with EcoRI-HF, and PacI for the double digestion as a positive control and attempt to integrate them in competent yeasts with RFP.
* Quantification of RFP expression in 3 different colonies with a plate reader.
* Sequencing of another promoter, ADH, on PCP\_Mxi1 plasmid (the one resulting from the gibson).

##### Week 8 (09/5/2016 – 09/11/2016)

* Pre-culture of yeasts with RFP to prepare the integration.
* Linearization and integration of c6\_TDH3\_1,2 and 3 in yeasts expressing RFP.
* Quantification of RFP expression with a plate reader in 3 different colonies (of yeast with the RFP integrated) and exclusion of the 2nd and 3rd due to poor/absent of expression (W303 yeasts were used as a blank and all cells were resuspended in PBS)
* Observation under a microscope (with filter for RFP) justify exclusion of colony 2 and 3 due the few fluorescent colonies.
* Inoculation and miniprep of c6\_TDH3\_1,2,3 and PCP\_Mxi1 for later integration in competent yeasts.
* Linearization of C6\_TDH3\_1,2,3 again (as the site used first was no good for integration in yeasts) with AsiSI and PacI restriction enzymes.
* Test of AsiSI enzyme.
* Linearization of TPGI\_tef1 for integration in yeasts with CYC\_GFP.
* Quantification of expression of GFP in yeasts with Cyc\_GFP and Tef1\_GFP (3 colonies from each).
* Inoculation of 3 new colonies from Tef1\_GFP plate as the previous ones didn’t show any expression of GFP.

##### Week 9 (09/12/2016 – 09/18/2016)

* Quantification of expression of GFP in yeasts with Cyc\_GFP and Tef1\_GFP (3 colonies from each).
* Preparation of SD agar plates lacking Leu, Ura and Trp (some have geneticin).
* Inoculation of 3 new colonies from Tef1\_GFP plate as the previous ones didn’t show any expression of GFP.
* Drop-out solution preparation and SD minimal medium plates.
* Observing colonies from Tef1\_GFP under microscope with a GFP filter and measuring GFP expression with a plate reader.
* Linearization and integration of C6\_TDH3\_1,2 and 3 plasmids.
* Inoculation of 10 colonies of Tef1\_GFP and overnight Plate-reader.
* Linearization of 1xPP7, 2xPP7, C6\_TDH3\_1,2,3, PCP\_Mxi1 and ADH1 plasmids, in order to perform integrations: 2 single integrations, 3 double and 2 triple.
* Inoculation and miniprep of ADH1 plasmid in 12 LB medium for later integrations.
* Design gBlocks (inserts) and primers needed to assemble the transistor (CYC1 promoter with scaffold gRNAs).

##### Week 10 (09/19/2016 – 09/25/2016)

* Inoculation and minipreps of ADH1 plasmid
* Overnight plate reader of W303 (as reference for the fluorescence), Tef1\_GFP, Tef1\_GFP+PCP\_Mxi\_Cas9+1xPP7, Tef1\_GFP+PCP\_Mxi\_Cas9+2xPP7, Tef1\_GFP+PCP\_Mxi\_Cas9+1xPP7 in GALACTOSE medium Tef1\_GFP+PCP\_Mxi\_Cas9+2xPP7 in GALACTOSE medium
* Integration of C6\_TDH3\_1,2,3 in yeasts containing RFP\_ADH1 and ADH1 plasmid in 3 colonies of cyc\_GFP.
* PCR purification of cyc\_c6\_1xPP7 and 2xPP7,and p404\_c6\_1xPP7 and 2xPP7.
* Gibson Assembly and transformation of p404\_c6\_1xPP7 with cyc\_c6\_1xPP7 and p404\_c6\_2xPP7 with cyc\_c6\_2xPP7.
* Gibson Assembly, transformations and PCR colony of p404\_c6\_1xPP7 with cyc\_c6\_1xPP7 and p404\_c6\_2xPP7 with cyc\_c6\_2xPP7 for tests on CYC promoter.

##### Week 11 (09/26/2016 – 10/02/2016)

* Plate reader to test activation and repression levels.
* 8 PCR were performed (including negative controls): On C6\_TDH3\_1,2,3 to substitute Kan resistance with the Ura selection marker. On cyc\_c6 two times , one with 1xPP7 and the second with 2xPP7 to target c6 region of CYC promoter for repression. On cyc\_c3 to obtain a plasmid containing the scaffold MS2 that targets c3 region of CYC promoter for Activation.
* PCR purification of previous PCR products.
* PCRs for C6\_TDH3\_1,2 (as they didn’t work the first time), for PCP\_Mxi1 and ADH1 to insert VP64 of ADH1 in PCP\_Mxi plasmid.for Ura selection marker to insert it in C6\_TDH3\_1,2,3.
* Gibson assembly and transformation of: C6\_TDH3\_1,2,3 with Ura (selection marker), p404 with cyc\_c6\_1xPP7 and CYC\_c3\_1xPP7 (test activation vs repression) PCP\_Mxi1+VP64.
* First [FACS](https://2016.igem.org/Team%3AEPFL/Protocols#facs)  of Tef1 promoter to test the repression by scTef1\_PP7 and scTef1\_2PP7 modules of Tef1 promoter.

##### Week 12 (10/03/2016 – 10/09/2016)

* Overnight plate reader to test the repression by scTef1\_PP7 and scTef1\_2PP7 modules of Tef1 promoter.
* Overnight plate reader to test the activation of CYC1 promoter by scCYC1\_c3 module.
* Flow cytometry analysis of CYC1 promoter to test the activation of CYC1 promoter by scCYC1\_c3 module.
* Flow cytometry analysis of CYC1 promoter to test repression by scCYC1\_PP7: GAL-inducible NOT gate
* Linearization and integration of C6\_TDH3\_1,2,3 with Uracil resistance in yeasts containing TDH3-RFP and dCAS9-ADH1 plasmids.

##### Week 13 (10/10/2016 – 10/16/2016)

* Second flow cytometry analysis of CYC1 promoter to test the activation of CYC1 promoter by scCYC1\_c3 module.
* Second flow cytometry analysis of CYC1 promoter to test repression by scCYC1\_PP7: GAL-inducible NOT gate.
* Linearization and integration of C6\_TDH3\_1,2,3 with Uracil resistance into yeasts containing TDH3-RFP and ADH1-dCas9.
* Linearization and integration of C6\_TDH3\_1,2,3 with Uracil resistance into w303 strains and into yeasts containing TDH3-RFP plasmid.

##### Week 14 (10/17/2016 – 10/20/2016)

* PCR of biobricks and PCR purification
* Restriction digest of PCR products (biobricks)
* Ligation of pSB1C3 with the inserts.

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