Lab Notebook

Documentation of our work.

June

In the beginning of June, we had a general lab introduction. As a first step, were ordered our chosen backbones pOxyR_gfp and GFP-X0 and started designing our experimental plan for the summer.

10.06. - 14.06.

  • Got liquid LB, LB plates, LB + ampicillin plates, YPD + adenine plates from D-BIOL shop.
  • Received plate of E. coli Nissle 1917 (hereafter referred to as Nissle) from Slack lab (ETH).
  • Received GFP-XO plasmid (bacterial stab) from Addgene.

17.06. - 21.06.

  • Preparation of ampicillin stocks, LB + ampicillin medium, YPD medium, and glycerol.
  • Autoclaving of materials.
  • Miniprep plasmid preparation of GFP-XO.
  • Made glycerol stocks of Nissle.
  • Received S. cerevisiae BY4743 (hereafter referred to as BY4743) from Frank van Drogen (ETH).

Additionally,
  • Held our first school workshop.
  • Started preparing the scripts and experiment material for the Wyss BioSTARS camp workshop.

24.06. - 28.06.

  • Received DNA fragments from TWIST: YAP1-Spel, YAP1-BamHI, pTRP1, UAS_pTRX2, TGA2_Spe_Bam, As1_CaMV35S, pGFP-polyL-pOxyR.
  • Received S. cerevisiae BY4741 (hereafter referred to as BY4741) from Zamboni lab (ETH). Made glycerol stocks of it.
  • Prepared MSG-URA liquid medium and plates. Genomic amplification of adh1 terminator (tadh1) from BY4743 via colony PCR using primers TADH1-FW and TRX2-Rv or CaMV35S-Rv. PCR clean-up using kit.
  • Restriction enzyme digestions of GFP-XO plasmid with BamHI and EcoRI. Purification using PCR clean-up kit.
  • Gibson assembly of digested GFP-XO backbone, tadh1 and UAS_pTRX2 or As1_CaMV35S.
  • Gibson product transformed into heat-shock competent E. coli DH5ɑ (“long” protocol).
  • Transformation success checked by restriction digest of plasmids obtained from colonies. Digests were checked by gel electrophoresis for expected fragment sizes. Obtained positive results for at least one colony of each plasmid.
  • Successfully cloned colonies were cultured and glycerol stocks prepared.

Additionally, we Held our second school workshop.
July

01.07. - 05.07.

  • Restriction enzyme digestion of CaMV35S-GFP with SpeI and BamHI. Purification using PCR clean-up kit.
  • Gibson assembly of digested CaMV35S-GFP with TGA2_SpeI_BamHI.
  • Gibson product transformed into DH5ɑ (“long” protocol).
  • Colonies were checked by restriction digest and gel electrophoresis. Obtained multiple positive results.
  • Restriction enzyme digestion of Trx2-GFP with SpeI and BamHI. Purification using PCR clean-up kit.
  • Gibson assembly of digested Trx2-GFP with YAP1-SpeI and YAP1-BamHI.
  • Gibson product transformed into DH5ɑ (“long” protocol).
  • One colony grew and was checked by restriction digest and gel electrophoresis. Cloning unsuccessful.
  • Sequencing of Trx2-GFP, Tga2-CaMV35S-GFP, CaMV35S-GFP plasmids. One Tga2-CaMV35S-GFP clone had mutation in the start codon. Stocks of that plasmid were discarded.
  • Trx2-GFP, Tga2-CaMV35S-GFP, CaMV35S-GFP plasmids successfully transformed into yeast BY4741 (LiAc-method). The used Tga2-CaMV35S-GFP was mutated, which was unknown at the time. Sequencing results came in just after. Transformations successful.
  • Attempted cloning of Yap1 fragments and Trx2-GFP directly into yeast.
  • Testing different settings for TECAN fluorescence measurements of yeast GFP constructs treated with H2O2. First TECAN fluorescence assay for yeast GFP constructs. Cells in liquid culture (MSG-URA/SC-MSG) at OD600=0.1, treated with H2O2.

08.07. - 12.07.

  • Checked colonies of Yap1-Trx2-GFP cloning into yeast by colony PCR and gel electrophoresis. Cloning unsuccessful.
  • TECAN fluorescence assay for yeast GFP constructs. Cells in liquid culture (MSG-URA/SC-MSG) at OD600=0.,1 treated with H2O2.
  • Restriction enzyme digestion of Trx2-GFP with SpeI and BamHI. Purification using PCR clean-up kit.
  • Gibson assembly of digested Trx2-GFP with YAP1-SpeI and YAP1-BamHI.
  • Gibson product amplified by PCR and checked by gel electrophoresis. Assembly unsuccessful.
  • Prepared heat-shock competent Nissle.
  • Tested “fast” transformation protocol on competent Nissle. Unsuccessful.
  • Transformation of BY4741 with correct (unmutated) Tga2-CaMV35S-GFP plasmids.

15.07. - 19.07.

  • Colonies from last week's BY4741 transformation checked by yeast colony PCR and gel electrophoresis. Transformations successful.
  • TECAN fluorescence assay for yeast GFP constructs. Single colonies resuspended in medium (MSG-URA/SC-MSG), treated with H2O2.
  • Restriction enzyme digestion of Trx2-GFP with XmaI and EcoRI (removal of Trx2, resulting in tadh1-GFP-XO). Gel purification.
  • Error prone PCR (epPCR) of Trx2 with different Mn-concentrations. Small volume of products checked by gel electrophoresis, the rest purified using PCR clean-up kit. Bands at expected sizes. EpPCR products inserted into digested plasmid (tadh1-GFP-XO) by Gibson assembly. EpPCR Gibson products amplified by PCR and checked by gel electrophoresis. Some plasmids were the correct size.
  • Test run of Wyss BioStars camp workshop experiments. Successful.
  • Generation of heat-shock competent DH5ɑ (CaCl2 method).

22.07. - 26.07.

  • Test transformation (“long” protocol) of newly prepared competent cells with GFP-XO. Successful.
  • Transformation of DH5ɑ with epPCR Trx2-GFP plasmids (“long” protocol). Colonies checked by colony PCR and sequencing. Sequencing revealed almost exclusively unmutated Trx2. EpPCR failed.
  • Received DNA fragments from TWIST: new_OxyR, new_YAP1_BamHI.
  • Restriction enzyme digestion of Trx2-GFP with SpeI and BamHI. Purification using PCR clean-up kit.
  • Gibson assembly of digested Trx2-GFP with YAP1-SpeI and new_YAP1_BamHI.
  • Gibson product transformed into DH5ɑ (“long” protocol). No colonies.
  • Repeated Restriction enzyme digestion of Trx2-GFP with SpeI and BamHI twice. Gel electrophoresis of both digests showed too large products. Purification using PCR clean-up kit and gel purification, respectively. Purification from gel produced very low yield.
  • Gibson assembly of digested and gel purified Trx2-GFP with YAP1-SpeI and new_YAP1_BamHI. Gibson product transformed into DH5ɑ (“long” protocol). No colonies.
  • Purified new Trx2-GFP plasmid from over night culture and tried digestions with different enzymes and combinations. BamHI was the only enzyme that did not function correctly.
  • Received OxyR-OxyS-GFP plasmid from Dr. Tsvetan Kardashliev (Univ. of Basel).
  • Transformation of DH5ɑ with OxyR-OxyS-GFP (“long” protocol). Successful.
  • Restriction enzyme digestion of OxyR-OxyS-GFP with NcoI and BsphI. Gel electrophoresis showed unexpected bands.
  • Tested “fast” transformation protocol with DH5ɑ and GFP-XO. Successful. Nissle from previous test not competent.

Additionally, we held the Wyss BioSTARS camp workshop!
August

29.07. - 02.08.

  • Tested different enzyme batches, concentrations and incubation times for Trx2-GFP BamHI digestion. Gel electrophoresis with SDS-containing loading dye as well as no-SDS dye. SDS lead to approximately expected band height. Gel purification of that band.
  • Gibson assembly of digested and gel purified Trx2-GFP with YAP1-SpeI and new_YAP1_BamHI. Gibson product transformed into DH5ɑ (“long” protocol). No colonies.
  • Sequencing of OxyR-OxyS-GFP plasmid (because of unexpected bands after digestion). Correct sequence. Repeated restriction enzyme digestion of Trx2-GFP with SpeI and BamHI (newly opened tube). Checked by gel electrophoresis. Wrong sizes.
  • Restriction enzyme digestion of Trx2-GFP with XmaI and EcoRI (removal of Trx2, resulting in tadh1-GFP-XO). Incorrect size, gel purified nonetheless.
  • EpPCR of Trx2. Subsequent DpnI digestion. Products purified using PCR clean-up kit.
  • EpPCR products inserted into digested plasmid (tadh1-GFP-XO) by Gibson assembly.
  • Transformation of DH5ɑ with epPCR Trx2-GFP plasmids (“fast” protocol). No colonies.
  • Repeated transformation of DH5ɑ with epPCR Trx2-GFP plasmids (“long” protocol). No colonies.
  • Backbone amplification PCR of OxyR-OxyS-GFP. Product checked by gel electrophoresis and purified from gel.
  • Gibson assembly of OxyR-OxyS-GFP backbone and new_OxyR (OxyR-J23101-PL-OxyS). Gibson product transformed into DH5ɑ (“medium fast” protocol). No colonies.
  • Repeated epPCR of Trx2 and subsequent DpnI digestion.

05.08. - 09.08.

  • Trx2 epPCR products (from last week) checked by gel electrophoresis. No bands.
  • Repeated test of restriction enzyme digestion of Trx2-GFP with SpeI. Checked by gel electrophoresis and gel purified.
  • Trx2-GFP SpeI digest was further digested with BamHI. Gel electrophoresis did not reveal any bands.
  • Tested Gibson assembly mix with positive control and transformed into DH5ɑ (“long” protocol). Successful.
  • Generation of heat-shock competent Nissle (CaCl2 method).
  • Competence of Nissle tested by transformation with GFP-XO (“long” protocol). Successful.
  • Restriction enzyme digestion of OxyR-OxyS-GFP with BspHI. Tried twice, both checked by gel electrophoresis. One reaction produced the correct band. This was purified from the gel.
  • Gibson assembly of OxyR-OxyS-GFP digest and new_OxyR (OxyR-J23101-PL-OxyS). Gibson product transformed into DH5ɑ and Nissle (“long” protocol). Tiny colonies of DH5ɑ, good colonies of Nissle. Nissle colonies checked by colony PCR and sequencing. Correct sequences for two colonies.
  • Restriction enzyme digestion of Trx2-GFP with XmaI and EcoRI (removal of Trx2, resulting in tadh1-GFP-XO). Incorrect size.
  • EpPCR of Trx2. Subsequent DpnI digestion. EpPCR products checked by gel electrophoresis.
  • Backbone amplification PCR of Trx2-GFP. Product checked by gel electrophoresis. Gel revealed the wrong size of PCR product. Repeated backbone amplification PCR of Trx2-GFP. Failed again. Did not continue with Trx2 epPCR cloning.
  • TECAN fluorescence assay of OxyR-OxyS-GFP and OxyR-J23101-PL-OxyS-GFP plasmids in Nissle. Overnight culture diluted in LB to OD600 = 0.5, treated with H2O2.
  • EpPCR of OxyR-J23101-PL-OxyS. Subsequent DpnI digestion. EpPCR products checked by gel electrophoresis. No bands.
  • Received mScarlet plasmid (bacterial stab) and cultured it.

12.08. - 16.08.

  • OxyR-J23101-PL-OxyS-GFP epPCR products from last Calendar Week remigrated on fresh gel due to suspicion of faulty gel. Still no bands.
  • Repeated epPCR of OxyR-J23101-PL-OxyS. Subsequent DpnI digestion.
  • EpPCR products checked by gel electrophoresis. No bands. Primer concentrations were incorrect.
  • Miniprep plasmid preparation of mScarlet.
  • Restriction enzyme digestion of mScarlet with EcoRI and SalI (Extracting mScarlet). Checked by gel electrophoresis and purified from gel.
  • Repeated epPCR of OxyR-J23101-PL-OxyS again. Subsequent DpnI digestion. EpPCR products checked by gel electrophoresis and purified from gel.
  • Transformation of OxyR-OxyS-GFP into Nissle (“medium fast” protocol). Successful.
  • Restriction enzyme digestion of Trx2-GFP and CaMV35S-GFP with EcoRI and SalI (Removing GFP for mScarlet insertion). Checked by gel electrophoresis and purified from gel.
  • Ligation of Trx2-GFP/CaMV35S-GFP with mScarlet. Ligations transformed into DH5ɑ (“fast” protocol). No colonies.
  • TECAN fluorescence assay of OxyR-J23101-PL-OxyS-GFP and OxyR-OxyS-GFP plasmids in Nissle and DH5ɑ.
  • Over night cultures resuspended in PBS and treated with H2O2. Failed to produce signal. Backbone amplification PCR of Trx2-GFP (longer elongation time). Checked by gel electrophoresis. PCR product too short.
  • Restriction enzyme digestion of Trx2-GFP and Tga2-CaMV35S-GFP with EcoRI and XmaI (for Gibson with epPCR promoters). Checked by gel electrophoresis and purified from gel.
  • TECAN fluorescence assay of OxyR-J23101-PL-OxyS-GFP and OxyR-OxyS-GFP plasmids in Nissle and DH5ɑ. Over night cultures diluted to OD600 = 0.5 in LB, treated with H2O2.
  • Tested growth of Nissle/DH5ɑ in M9 medium. No growth.
  • TECAN fluorescence assay of OxyR-J23101-PL-OxyS-GFP and OxyR-OxyS-GFP plasmids in Nissle and DH5ɑ. Over night cultures diluted to OD600 = 0.5, resuspended in M9, treated with H2O2.
  • Backbone amplification PCR of OxyR-OxyS-GFP with new primers. Checked by gel electrophoresis and purified from gel. Low yield.
  • EpPCR of OxyS. Subsequent DpnI digestion. EpPCR products purified using PCR clean-up kit.

19.08. - 23.08.

  • Gibson assembly of Trx2 and Tga2 epPCR products with their respective backbones (digests from last week). Gibson products transformed into Nissle (“long” protocol). No colonies.
  • Sequencing of OxyR-J23101-PL-OxyS-GFP plasmid. Correct sequence.
  • Backbone amplification PCR of Trx2-GFP with new primers. Checked by gel electrophoresis. PCR product too short.
  • Gibson assembly of OxyR-OxyS-GFP backbone (last week) and OxyS epPCR products. Gibson products transformed into DH5ɑ and Nissle (“long” protocol). No colonies.
  • Restriction enzyme digestion of Trx2-GFP and CaMV35S-GFP with EcoRI and SalI (Removing GFP for mScarlet insertion). Checked by gel electrophoresis. Incorrect sizes.
  • TECAN fluorescence assay of OxyR-J23101-PL-OxyS-GFP and OxyR-OxyS-GFP plasmids in Nissle and DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in M9, treated with H2O2.
  • TECAN fluorescence assay of OxyR-J23101-PL-OxyS-GFP and OxyR-OxyS-GFP plasmids in Nissle and DH5ɑ. Over night cultures diluted to OD600 = 0.8, resuspended in PBS, treated with H2O2.
  • Sequencing of Trx2-GFP backbone amplification. Incomplete sequence.
  • Repeated restriction enzyme digestion of Trx2-GFP and CaMV35S-GFP with EcoRI and SalI (Removing GFP for mScarlet insertion). Checked by gel electrophoresis and purified from gel.
  • Ligation of Trx2-GFP with mScarlet. Ligations transformed into DH5ɑ (“fast” protocol). Multiple colonies grew.
  • Backbone amplification PCR of OxyR-OxyS-GFP with new primers. Checked by gel electrophoresis and purified from gel.
  • Repeated Gibson assembly of OxyR-OxyS-GFP backbone (this week) and OxyS epPCR products. Gibson products transformed into DH5ɑ (“long” protocol). Multiple colonies grew.
  • Restriction enzyme digestion of pDRF1-GW ymScarletI with EcoRI and SalI (Extracting mScarlet). Checked by gel electrophoresis and purified from gel.
  • Repeated backbone amplification PCR of Trx2-GFP (fresh plasmid) with new primers. Checked by gel electrophoresis. PCR product too short.

26.08. - 30.08.

  • Colonies from Trx2-mScarlet (ligation and transformation last week) checked for mScarlet fluorescence in TECAN plate reader. Only background signal.
  • Two Trx2-mScarlet colonies were sequenced. Positive sequencing results.
  • (2x) Restriction enzyme digestion of GFP-XO and Tga2-CaMV35S-GFP with EcoRI and SalI (to remove GFP). Checked by gel electrophoresis and purified from gel.
  • Ligation of digested GFP-XO and digested Tga2-CaMV35S-GFP with mScarlet. Ligations transformed into DH5ɑ (“medium fast” protocol). Colonies grew. Colonies of Tga2-CaMV35S-mScarlet and mScarlet-XO sent to sequencing. Unsuccessful reads.
  • TECAN fluorescence assay of all OxyR-epOxyS-GFP variants in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • Received E. coli K12, Pseudomonas protegens Pf-5, Pseudomonas putida 1243 and P. alloputida mt-2 kt2440 (hereafter referred to as K12, Pf-5, P. putida 1243 and P. putida 1320 respectively) from Zamboni lab (ETH).
  • Generation of electro-competent K12, Pf-5, P. putida 1243 and P. putida 1320 with cold water. Electroporation of K12, P. putida 1243 and P. putida 1320 with OxyR-OxyS-GFP. Plenty of colonies for K12, none for Pf-5 and P. putida strains.
  • Repeated transformation for Pf-5 and P. putida strains. No colonies of Pf-5. Colonies grew from both P. putida strains.
  • Generation of electro-competent Pf-5 with MOPS.
  • Electroporation of Pf-5 with OxyR-OxyS-GFP. Few colonies.
  • Sequencing of OxyR-OxyS-GFP transformed K12, P. putida 1243 and P. putida 1320 and Pf-5. Positive results for K12 and P. putida strains. No good reads for Pf-5.
  • (2x) Restriction enzyme digestion of OxyR-OxyS-GFP with EcoRI. Checked by gel electrophoresis and purified from gel. Low yields.
  • TECAN fluorescence assay of OxyR-epOxyS-GFP variants in DH5ɑ. Over night cultures grown to OD600 = 1.24, resuspended in PBS, treated with H2O2.

September

02.09. - 06.09.

  • OxyR-OxyS-GFP from Pf-5 colonies sent to sequencing again. No good reads. Colonies of Tga2-CaMV35S-mScarlet and mScarlet-XO from last Calendar Week sent to sequencing again. Correct sequence for mScarlet-XO, none for Tga2-CaMV35S-mScarlet.
  • Ligation of digested GFP-XO and digested Tga2-CaMV35S-GFP with mScarlet. Ligations transformed into DH5ɑ (“medium fast” protocol). Colonies grew and were sent to sequencing. Positive sequencing result for mScarlet-XO and Tga2-CaMV35S-mScarlet.
  • Transformations of BY4741 with pDRF1-GW ymScarletI, Tga2-CaMV35S-mScarlet, mScarlet-XO and Trx2-mScarlet. Colonies grew. BY4741 colonies were treated with H2O2 and checked for mScarlet fluorescence in TECAN plate reader. Only signal for pDRF1-GW ymScarletI. Cells likely died due to high H2O2 concentration.
  • Restriction enzyme digestion of OxyR-OxyS-GFP with EcoRI. Checked by gel electrophoresis and purified from gel.
  • Genomic amplification PCR of Catalase (KatG) from K12 colony. Checked by gel electrophoresis and purified from gel.
  • Gibson assembly of digested OxyR-OxyS-GFP and catalase from genomic amplification.
  • Gibson product transformed into DH5ɑ (“long” protocol). Colonies checked by sequencing. Positive results.
  • Backbone amplification PCR of OxyR-OxyS-GFP with new primers. Checked by gel electrophoresis and purified from gel.
  • Restriction enzyme digestion of Trx2-GFP with XbaI and BamHI. Checked by gel electrophoresis. Wrong size.
  • (3x) Genomic amplification PCR of Yap1 from BY4741 colony with new primers. Different PCR settings. Checked by gel electrophoresis. Obtained correct size in the end, but very low yield.
  • TECAN fluorescence assay of mScarlet constructs in BY4741. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2. Almost no mScarlet emission, even in mScarlet-XO.
  • TECAN fluorescence assay of OxyR-OxyS-GFP in K12, Nissle, DH5ɑ and OxyR-OxyS-KatG-GFP in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • Sent mScarlet-XO to sequencing. Positive result.
  • First setup of intracellular ROS measurement in DH5ɑ with OxyR-OxyS-GFP and OxyR-OxyS-KatG-GFP. No usable data.
  • Received dmOxyS_Og and dmOxyS_BS DNA fragments from IDT.
  • Gibson assembly of OxyR-dmOxyS_BS0-GFP and EcoRI digested OxyR-OxyS-GFP. Wrong, should have been OxyR-OxyS-GFP backbone amplification.
  • Gibson product transformed into DH5ɑ (“long” protocol). Colonies grew. Contained only backbone.
  • TECAN fluorescence assay of OxyR-epOxyS-GFP variants in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.

09.09. - 13.09.

  • Sequencing of two OxyR-epOxyS-GFP plasmids.
  • Transformation of OxyR-OxyS-KatG-GFP into Nissle (“long” protocol). Colonies checked by sequencing. Positive results.
  • (2x) Genomic amplification PCR of Yap1 from BY4741 colony with new primers. Checked by gel electrophoresis. Obtained the correct size in the end. Purified from gel.
  • (3x) Backbone amplification PCR of Trx2-GFP. Trying different PCR settings. Checked by gel electrophoresis. Finally obtained band with correct size, but concentration extremely low.
  • Tried PCR amplification of low yield Trx2-GFP backbone. Checked by gel electrophoresis. Wrong size.
  • Restriction enzyme digestion of OxyR-OxyS-GFP with EcoRI. Checked by gel electrophoresis and purified from gel.
  • Transformation of BY4741 with mScarlet-XO. Colonies grew.
  • Backbone amplification PCR of OxyR-OxyS-GFP with new primers. Checked by gel electrophoresis and purified from gel.
  • Gibson assembly of OxyR-OxyS-GFP (from backbone amplification) with dmOxyS_BS0 – dmOxyS_BS7 and dmOxyS_Og0 – dmOxyS_Og9 and dmOxyS_BSnoBGmut8. Gibson products transformed into DH5ɑ (“long” protocol). Colonies checked by sequencing. Correct sequences for BS0 – 4, Og1 – 7.
  • TECAN fluorescence assay of OxyR-OxyS-GFP in P. putida 1243 & 1320. Over night cultures diluted to OD600 = 0.5, resuspended in PBS, treated with H2O2.
  • TECAN fluorescence assay of OxyR-OxyS-GFP in Nissle, Pf-5, P. putida 1243 & 1320. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • TECAN fluorescence assay of GFP constructs in BY4741. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • Restriction enzyme digestion of GFP-XO with EcoRI. Checked by gel electrophoresis and purified from gel. PCR amplified mScarlet from pDRF1-GW ymScarletI using new primers to reduce distance to promoter in final construct. PCR checked by gel electrophoresis and product purified from gel.
  • Gibson assembly of digested GFP-XO and mScarlet (from PCR). Gibson product (mScarlet-XO V2) transformed into DH5ɑ (“long” protocol).

16.09. - 20.09.

  • mScarlet-XO-v2 colonies from last Calendar Week sent for sequencing. Positive results.
  • (2x) TECAN fluorescence assay of OxyR-dmOxyS_Og-GFP variants in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • TECAN fluorescence assay of OxyR-dmOxyS_BS-GFP variants in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • TECAN fluorescence assay of OxyR-epOxyS-GFP variants in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • (4x) Backbone amplification PCR of Trx2-GFP. Trying different PCR settings. Checked by gel electrophoresis. Mainly wrong sizes. Eventually extracted band of correct size.
  • Gibson assembly of Trx2-GFP (from backbone amplification) and Yap1 (From genomic amplification last week). Gibson product transformed into DH5ɑ (“fast” protocol).

23.09. - 27.09.

  • BY4741 was transformed with mScarlet-XO V2. Colonies grew.
  • TECAN fluorescence assay of OxyR-dmOxyS_OG-GFP variants in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2.
  • TECAN fluorescence assay of OxyR-OxyS-GFP and OxyR-OxyS-KatG-GFP in DH5ɑ. Over night cultures diluted to OD600 = 1, resuspended in PBS, treated with H2O2. 20 ul 100 mM H2O2 added again after 2 h.
  • Technical error in the TECAN. Repeated the experiment with same settings. A single set of fluorescence measurements was made for mScarlet-XO V2 in BY4741. V2 does produce mScarlet fluorescence.