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 of cultures with the plasmids(all of them).
• We received from IDT the eight inserts (gBlocks); gBlocks were resuspended in TE buffer.
• 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 of the whole PCR products and quantification with Nanodrop®.
• First try of Gibson assembly for 8 of 9 plasmids needed.

Week 3 (08/01/2016 – 08/07/2016)

• 1L of LB agar medium (50 plates) with ampicillin .
• 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 of TDH3+RFP and pGal1+C6_TDH3_3.
• PCR colony 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 (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 of TEF1_GFP (repression) integration in Yeasts.
• PCR purification of TPGI and tef1.
• Gibson Assembly, transformation, PCR colony and inoculation of scRNA_Trp+tef1.
• Replate 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 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.