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®.
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.
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.
