iGEM 2024 — Team eth-zurich

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Notebook
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{% block title %}Lab Notebook{% endblock %}
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June

July

August

September

# 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.

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Engineering
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{% block title %}
Engineering
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{% block lead %}
Our Design-Build-Test-Learn process.
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{% block page\_content %}

# The choices behind our construct and project designs, and how much we have achieved.

Introduction

Species and strain choice

Bacterial plasmids design

Yeast plasmids design

Engineering process

Primer sequences

## Introduction

With Reactive oxygen Species at the center of many diseases, harnessing them as a marker would
enable innovative and broad research, diagnostics and therapeutic avenues. We have, thus, designed a
cross-species ROS-detection platform aimed at detecting harmful ROS concentrations which naturally
occur in diseases. Such a sensor could be paired with a detection and/or therapeutic output
depending on the aim. As a proof of concept, we chose to focus on 2 diseases, each highly
cumbersome: IBD in humans, and fireblight in plants. (see [Description](description) for more
details)

## Species and strain choice

Species choice is crucial since each organism presents different intrinsic ways of coping with ROS
and oxidative stress and will, therefore, harbor a specific intracellular environment (pH, redox,
electric, temperature variations…). These intrinsic factors will affect the platform’s
characteristics once inserted. Within the same strain, even growth conditions will affect the
system, but here we need to focus on the most important variables (ie. species, strain). Since our
main work is to tune expression, it is important from the beginning to select a host which will be
as close as possible to the end application host.

* For our medical case-study the choice was easy, as E. coli Nissle 1917 is the most engineered
  microbiota strain used in several studies for similar chronic inflammation therapies in the
  gut(1, 2). Comparison with classic lab-strains like E. coli DH5a and K-12 would be relevant to
  assess the importance of strain variability on the oxidative stress response.
* For our agritech application, we considered all the main bacterial biocontrol agents as
  potential vectors. Indeed, many currently used biocontrol sprays are [bacteria-based](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9502092/#:~:text=Streptomyces%20spp.%20is%20one%20of,fungal%20plant%20diseases%20%5B70%5D)
  (Pseudomonads and Erwiniaceae), like Pseudomonas fluorescens A506, EPS62e, Pseudomonas protegens
  Pf5 or Pantoea agglomerans/vagans E325(3–5). At the suggestion of Cosima Pelludat and Theo
  Smits, we looked into Pantoea vagans C9-1 “white” strain, which displays interesting
  characteristics(4, 6). Its multiple [metabolic deficiencies](https://academic.oup.com/femsle/article/308/1/48/513658)
  restrict its lifespan in nature to a
  few days, alleviating biosafety concerns regarding GMOs and their dissemination. However, its
  carotenoid deficiency could impact its resistance to UV. All these strains would have been
  relevant to use as vectors, and we hope to be able to test them all with more time and
  collaborations. For now, we only managed to get P. protegens Pf5.
* Pseudomonas putida strains are used for biocontrol and many bioprocesses, showing strong
  tolerance of high ROS levels(7, 8). Therefore, we chose to include P. alloputida mt-2 kt2440 to
  our study, provided by the lab.

## Bacterial plasmids design

### oxyR-oxyS-gfp and oxyR-J23101-PL-oxyS-gfp

#### The reasons behind the design

This plasmid comes from the Panke lab, and was provided by Dr. Tsvetan Kardashliev. The native oxyRS
regulon drives expression of oxyR in one direction, and of oxyS in the other. In the Panke plasmid,
oxyS was replaced by the gfp gene.
In this instance, oxyR negatively regulates its own expression, so a GFP peak will naturally
decrease shortly, when oxyR stops being produced (through negative feedback). However, we want a
stable expression of our output protein.
To bypass oxyR’s impact on its regulation, we redesigned the promoter region to include a
constitutive promoter for oxyR (part BBa\_J23101 Berkeley 2006) and the sequence was adjusted next to
the oxyR gene as done in BBa\_K1104200 (NYMU-Taipei 2013). A polylinker (referred to as PL) was added
between the new promoter (BBa\_J23101) and the oxyRS regulon (named oxyS promoter), as shown in Figure 1.

![oxyR plasmid design](https://static.igem.wiki/teams/5356/description/oxyr.png)

**Figure 1. oxyR-J23101-PL-oxyS-gfp plasmid detail.**

#### Cloning design

The goal was to order synthetic DNA for the whole promoter region (J23101-PL-oxyS, see sequence in
the
Twist table below) but due to lab changes, we had to actually order a way larger fragment
encompassing
also oxyR and half of the GFP where ideal restriction sites were positioned (NcoI and BspHI). Due to
issues with enzymes, the ligation did not work out and we had to amplify the whole backbone from the
BspHI homology region to NcoI homology region (see Engineering details below).

### oxyR-dmoxyS\_**OG**-gfp and oxyR-dmoxyS\_**BS**-gfp

#### The OG plasmids explanation behind the mutations

oxyR-dmoxyS\_**OG**-gfp plasmids are the oxyR-oxyS-gfp but with designed mutations in the
binding site
region. These mutations were predicted computationally to affect binding more or less strongly (see
[Model page](model)).
However, we cannot predict beforehand the impact on the phenotype and ROS-sensing response in vivo.
Therefore, we tested several combinations of predicted mutations (variation in number, association
of strong and soft, different positions).
Here is a table of designed sequences (from the most mutated to the least):

| dmoxyS\_OG0 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacTATcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| --- | --- |
| dmoxyS\_OG1 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatCGggataatcgtTcattgctTtTctacctatcgccatgaacACTcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG2 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatCGggataatcgtTcattgctAtTctacctatcgccatgaacTATcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG3 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctTtTctacctatcgccatgaacTATcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG4 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacAATcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG5 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacTCTcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG6 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctTtTctacctatcgccatgaacTCTcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG7 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctTtTctacctatcgccatgaacACTcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG8 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtGctacctatcgccatgaacTATcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_OG9 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacTAAcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |

#### Understanding the aim behind the duplication

For oxyR-dmoxyS\_**BS**-gfp, however, we duplicated the binding site region (BS). Why? If you look
at this
scheme (Figure 2), you can see that the oxyR binding site regulates the expression of two promoters.
There is a **positive** regulation of the gfp gene, since the binding happens just before the
**-35 region
where the polymerase binds**, whereas it **negatively** regulates oxyR because the binding
happens
**between
the -35 and -10 regions, interfering with the transcription complex binding.**

![original sequence](https://static.igem.wiki/teams/5356/description/og-region.png)

**Figure 2. Original version of the oxyR-oxyS-GFP plasmid.**

We, thus, designed a version where there are 2 distinct oxyR binding regions (BR), as represented Figure 3. That way, we can
try and alleviate negative feedback on the oxyR gene expression with strong mutations in BR2, and
choose soft mutations for BR1 regulating GFP expression, in the hopes of tuning the sensing to
high-ROS levels. It is also possible that BR1 promotes oxyR expression since it is now in a
favorable position compared to the oxyR -35 site.

Since we never know what happens in vivo, we have
designed 9 versions, testing various mutation **strengths, numbers and combinations.**

![BR-duplicated version](https://static.igem.wiki/teams/5356/description/bs-region.png)

**Figure 3. Binding-region duplicated version of the oxyR-oxyS-GFP plasmid.**

Here is a table of designed sequences (from the most mutated to the least):

|  |  |  |  |
| --- | --- | --- | --- |
| Only with the 4 background mutations : 133(GC), 149(CG), 157(CG), 170(AT) | | | |
| dmoxyS\_BS0 | gaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacTATcgaatgcttgatagTgataatCgtTTattgCtatTctaGctatcgccatgaGCtaTcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| Additional soft 75(AT) 95(AT) 116(TA) 117(AT) and/or strong 140(GC) 143(TA) 171(TA) 174(TA) mutations | | | |
| dmoxyS\_BS1 | gaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatCGggataatcgtTcattgctTtTctacctatcgccatgaacACTcgaatgcttgatagTgataatGgtGTattgCtatTctaGctatcgccatgaGGtaGcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_BS2 | gaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctTtTctacctatcgccatgaacAATcgaatgcttgatagTgataatCgtTTattgCtatTctaGctatcgccatgaGCtaGcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_BS3 | gaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacAATcgaatgcttgatagTgataatCgtTTattgctatTctaGctatcgccatgaGCtaGcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_BS4 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacTATcgaatgcttgatagTgataatCgtGTattgctatTctaGctatcgccatgaGCtaGcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_BS5 | gaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctTtTctacctatcgccatgaacAATcgaatgcttgatagTgataatCgtTTattgCtatTctaGctatcgccatgaGCtaTcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_BS6 | gaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacAATcgaatgcttgatagTgataatCgtTTattgCtatTctaGctatcgccatgaGCtaTcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_BS7 | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatAGggataatcgtTcattgctAtTctacctatcgccatgaacTATcgaatgcttgatagTgataatCgtTTattgCtatTctaGctatcgccatgaGCtaGcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| dmoxyS\_BS8 | gaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatCGggataatcgtTcattgctTtTctacctatcgccatgaacACTcgaatgcttgatagGgataatGgtGCattgCtatTctaCctatcgccatgaAGtaGcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |

#### Cloning design

All plasmids were obtained by Gibson Assembly (backbone amplification from GFP N-ter and oxyR N-ter).

### oxyR-oxyS-katG-gfp

For the medical application, we chose to test the influence of catalase-expression on H202-induced
stress and response, since the ultimate goal would be to counteract elevated levels of ROS in
chronic inflammation. We aimed to use an intracellular H2O2 titration assay but planned to keep a
fluorescent reporter in case it did not work (good call), so we added the catalase gene just before
the GFP.
We would expect GFP levels to soar less with katG than without, since the catalase would help the
bacteria counter the stress more efficiently. H202 would therefore be degraded quicker, leading to
oxyR being less activated and thus GFP less expressed.
Note: GFP would be naturally less expressed since it is the second gene on the RNA.

#### Cloning design

The catalase gene in E.coli is katG, so we decided to genomically amplify it from our bacteria and
clone it before the GFP through Gibson Assembly (parallel amplification of the backbone).

## Yeast plasmid design

Yeast plasmids were based on the plasmid GFP-X0 we got from Addgene #115565 suited for **bacterial
expression (cloning phase)**
and **yeast expression (sensor-testing phase)**. In yeast, the strong
constitutive promoter adh1 induces expression of GFP, followed by the cyc1 terminator.

### CaMV35S-gfp and trx2-gfp

To test TGA2- and yap1- mediated sensing, we had to add them to the GFP-X0, where GFP would be the
**reporter protein**. The **TF-regulated promoter should regulate GFP** and not adh1 anymore,
so an
adh1-compatible terminator was added to close the ORF (padh1-tadh1), and added the CaMV35S/Trx2
promoter after (in front of the gfp gene, see Figure 4 below). However, we first added the adh1
terminator and the corresponding promoter, so that way we would obtain a plasmid which could give
the background activation due to native yap1 or cellular environment.

#### Cloning design

We genomically amplified tadh1 with 2 PCRs, one for CaMV35S homology and the other for trx2 homology.
We ordered the 2 promoters through Twist (see sequences in the Twist table below), and then did a
2-fragment Gibson Assembly (the terminator, the promoter, and the digested backbone on EcoRI+BamHI).

### tga2-CaMV35S-gfp and yap1-trx2-gfp

Tga2 and yap1 were ordered with Twist and cloned through Gibson Assembly (SpeI+BamHI sites). Where
tga2
worked on the first try, we never managed to integrate yap1. Since yap1 was too long for synthesis,
it
was separated into 2 parts. We thought maybe the homology between the 2 was too short, but ordering
a
new sequence did not work. We also have trouble preparing the backbone, with apparent restriction
enzyme
binding to the plasmid (higher bands than the plasmid), and switched to PCR amplification (see [Results](results)).

![tga2-yap1](https://static.igem.wiki/teams/5356/description/tga2-yap1.png)

**Figure 4. tga2-CaMV35S-gfp and yap1-trx2-gfp plasmids.**

### mScarlet versions of adh1-TGA2-tadh1-CaMV35S-GFP, adh1-tadh1-trx2-GFP and GFP-X0

Since the autofluorescence of yeast was impeding interpretation of fluorometric assays, we aimed to
replace the GFP genes by the mScarlet gene (600 nm emission peak, also where plants do not
autofluoresce).

#### Cloning design

We found a plasmid for ymScarletI expression with compatible restriction sites on Addgene called
pDRF1-GW ymScarletI, and we planned for ligation.

#### mScarlet-XO V2

However, no expression was noticed, we thought it might be because the distance between the
transcription start and translation start was longer for this design (also translation end). For
GFP-X0, we thus designed primers for Gibson Assembly, in order to preserve the same spacing in the
mScarlet-X0 as in the current GDP-X0 (between the adh1 promoter and cyc1 terminator) and managed to
obtain mScarlet expression. Running out of time, we did not have time to clone mScarlet the same way
in the other plasmids.

### epPCR plasmids

We aim to amplify the promoters (CaMV35s, Trx2, oxyS, J23101-PL-oxyS) of all our key plasmids through
error-prone PCR to obtain random mutagenesis of these regions. Flow cytometry would be then used for
highthrough-put screening of mutants (GFP+ or GFP- screenings) to select those with the best
ROS-detection range.
The plasmids were designed to keep restriction sites between each genetic element, so we first
tested Gibson Assembly of the epPCR library with XmaI+EcoRI digest of the plasmid. There was very
low efficiency, therefore, we also tried backbone amplification from EcoRI (end of promoter) to XmaI
(beginning) (see [Results](results)).

| oxyR-J23101-PL-oxyS-gfp | gagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagctcttctcctttgctcatctagtatttctcctctttaatctctagtatttctcctctttctctagtaaacgacgccggtatgtttctaatatgttacatattatcggggacattgttccagacaacaatctgttttattcaataggtatgatagtcgcaggctttggGAATTCGAGCTCGGTACCCGGGcATCCTaTAGAGTCGACCTGCAGGCATGCAAGCTTGGtttacagctagctcagtcctaggtattatgctagctactagagaaagaggagaaaatcgatatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgccacgttagccagccgacgcttagcgggcaaattcgtaagctggaagatgagctgggcgtgatgttgctggagcggaccagccgtaaagtgttgttcacccaggcgggaatgctgctggtggatcaggcgcgtaccgtgctgcgtgaggtgaaagtccttaaagagatggcaagccagcagggcgagacgatgtccggaccgctgcacattggtttgattcccacagttggaccgtacctgctaccgcatattatccctatgctgcaccagacctttccaaagctggaaatgtatctgcatgaagcacagacccaccagttactggcgcaactggacagcggcaaactcgattgcgtgatcctcgcgctggtgaaagagagcgaagcattcattgaagtgccgttgtttgatgagccaatgttgctggctatctatgaagatcacccgtgggcgaaccgcgaatgcgtaccgatggccgatctggcaggggaaaaactgctgatgctggaagatggtcactgtttgcgcgatcaggcaatgggtttctgttttgaagccggggcggatgaagatacacacttccgcgcgaccagcctggaaactctgcgcaacatggtggcggcaggtagcgggatcactttactgccagcgctggctgtgccgccggagcgcaaacgcgatggggttgtttatctgccgtgcattaagccggaaccacgccgcactattggcctggtttatcgtcctggctcaccgctgcgcagccgctatgagcagctggcagaggccatccgcgcaagaatggatggccatttcgataaagttttaaaacaggcggtttaaactagtcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaggggtccccaataattacgatttacgtatttaaatgaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttggggtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatcatgagggaagcggtgatcgccg |
| --- | --- |
| Yap1\_BamHI | gaattttgttctaaaatgaatcaagtgtgtggcactagacaatgtccaattccaaaGaaaccaatttctgctttggataaagaagtgtttgcttcttcttcAattttgtcttctaattctccagctttgactaatacttgggaatctcattctaatattactgataatactccagctaatgtgattgctactgatgctactaaatatgaaaattctttttctggctttggcagattgggctttgatatgtctgctaatcattatgtggtgaatgataattctacAggctctactgattctactggctctactggcaataaGaataaGaaaaataataataattctgatgatgtgttgccatttatttctgaatctccatttgatatgaatcaagtgactaatttCttttctccaggctctactggcattggcaataatgctgcttctaatactaatccatctttgttgcaatcttctaaagaagatattccatttattaatgctaatttggcttttccagaCgataattctactaatattcagttgcaaccattttctgaatctcaatctcaaaataaatttgattatgatatgttCtttagagattcttctaaagaaggcaataatttgtttggcgaatttttggaagaCgaCgaCgatgaCaaGaaagctgctaatatgtctgatgaCgaatcttctttgattaaaaatcagttgattaatgaagaaccagaattgccaaaacaatatttgcaatctgtgccaggcaatgaatctgaaatttctcaaaaGaatggctcttctttgcaaaatgctgataaaattaataatggcaatgataatgaCaatgataatgatgtggtgccatctaaagaaggctctttgttgagatgttctgaaatttgggatagaattactactcatccaaaatattctgatattgatgtggatggcttgtgttctgaattgatggctaaagctaaatgttctgaaagaggcgtggtgattaatgctgaagatgtgcagttggctttgaataaacacatgaattaaggatcctaataagcgaatttc |
| new\_Yap1\_BamHI | GaacaccataggtcagagtagtgacaagtgttggccatggaacaggtagttttccagtagtgcaaataaatttaagggtgagttttccgtttgtagcatcaccttcaccctctcctctgacagaaaatttgtgcccattaacatcaccatctaattcaacaagaattgggacaactccagtgaaaagttcttctcctttgctcatatgttaaatctcctgctaattaaaaagtaaagtgtgaattctgctgtgagcaattatcagtcagaatgcttgatCGggataatcgtTcattgctAtTctacctatcgccatgaacTATcgtggcgatggaggatggataatgaatattcgtgatcttgagtacctggtggcattggctgaacaccgccattttcggcgtgcggcagattcctgc |
| yap1-Spel | ccaagctggccgctctagaactagtatgtctgtgtctactgctaaaagatccttggatgtggtgtctccaggctctttggctgaatttgaaggctctaaatccagacatgaCgaaattgaaaatgaacatagaagaactggcactagagatggcgaagattctgaacaaccaaaGaaGaaaggctctaaaacttctaaGaaacaagatttggacccagaGactaaGcaaaaGagaactgctcaaaatagagcAgctcaaagagcttttagGgaaagGaaGgaaagaaaGatgaaagaattggaaaaGaaagtgcaatctttggaatctattcaacaGcaaaatgaagtggaagctacttttttgagagatcagttgattactttggtgaatgaattgaaGaaatatagaccagaaactagaaatgattctaaagtgttggaatatttggctagaagagatccaaatttgcatttttctaaaaataatgtgaatcattctaattctgaaccgattgatactccaaatgaCgatattcaagaaaatgtgaaacaaaaGatgaattttacttttcaatatccattggataatgataatgaCaatgataattctaaaaatgtgggcaaacagttgccatctccaaatgatccatctcattctgctccaatgccaattaatcaaactcaaaaGaaattgtctgatgctactgattcAtcttcAgctactttggattcAttgtctaaCtctaatgatgtgttgaaCaatactccaaattcAtctacttctatggattggttggataatgtgatttatactaatagatttgtgtctggcgaCgatggctctaattctaaaactaaaaatttggattctaatatgttttctaatgattttaattttgaaaatcaatttgatgaacaagtgtctgaattttgttctaaaatgaatc |
| TGA2\_SpeI\_BamHI | CCAAGCTGGCCGCTCTAGAACTAGTATGGCTGATACTTCTCCAAGAACTGATGTGTCTACTGATGATGATACTGACCATCCAGATTTGGGCTCTGAAGGCGCTTTGGTGAATACTGCTGCTTCTGATTCTTCTGATAGATCTAAAGGCAAAATGGATCAAAAAACTTTGAGAAGATTGGCTCAAAATAGAGAAGCTGCTAGAAAATCCAGATTGAGAAAAAAAGCCTATGTGCAACAGTTGGAAAATTCCAGATTGAAATTGACTCAGTTGGAACAAGAATTGCAAAGAGCTAGACAACAAGGCGTGTTTATTTCTGGCACTGGCGATCAAGCTCATTCTACTGGCGGCAATGGCGCTTTGGCTTTTGATGCTGAACATTCCCGTTGGCTGGAAGAAAAAAATAAACAAATGAATGAATTGAGATCTGCTTTGAATGCTCATGCTGGCGATTCTGAATTGAGAATTATTGTGGATGGCGTGATGGCTCATTATGAAGAATTGTTTAGAATTAAATCTAATGCTGCTAAAAATGATGTGTTTCATTTGTTGTCTGGCATGTGGAAAACTCCAGCTGAAAGATGTTTTTTGTGGTTGGGCGGCTTTAGATCTTCTGAATTGTTGAAATTGTTGGCTAATCAGTTGGAACCAATGACTGAAAGACAGTTGATGGGCATTAATAATTTGCAACAAACTTCTCAACAAGCTGAAGATGCTTTGTCTCAAGGCATGGAATCTTTGCAACAATCTTTGGCTGATACTTTGTCTTCTGGCACTTTGGGCTCTTCTTCTTCTGGCAATGTGGCTTCTTATATGGGCCAAATGGCTATGGCTATGGGCAAATTGGGCACTTTGGAAGGCTTTATTAGACAAGCTGATAATTTGAGATTGCAAACTTTGCAACAAATGATTAGAGTGTTGACTACTAGACAATCTGCTAGAGCTTTGTTGGCTATTCATGATTATTTTTCCAGATTGAGAGCTTTGTCTTCTTTGTGGTTGGCTAGACCAAGAGAAGGATCCTAATAAGCGAATTTC |
| pTRP1 | CTCCTATGCACATATATTAATTAAAGTCCAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATATTTCGGATGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTCCAAGCTGGCCGCTCTAGAACTAGTATGTCTGTGTCTACTGCTAAAAG |
| UAS\_pTRX2 | CAAATGCCTGCAAATCGCTCCCCCCGGGCCTTTCAGATCTGTCATCTTGAGTGCCAGTGAATATTAAGTAATCATCAAAGTATATGTGTAATTGTTTATACTCTTAGTAAAGGATGCTCCCTACAAGGTGGCTCTTTTCTTACTAAGCGCGTTCAGTTTCCAGCCAGCCGAAAGAGGGATATCAGTATATAAGAAAGCCATTCGGGGGATGAAAAGCTGACAAGAGAATAACGAGGACCAGTTTTTATTTGTTGTCTAGCAAGAATTATACACGCACACATACACGAGAGTCTACGATATCTTTAAATAACACATCAATAAGCAGGAATTCATGAGTAAAGGAGAAG |
| As1\_CaMV35S | CCTGCAAATCGCTCCCCCCGGGCAGCTTCTGACGTAAGGGATGACGCACCTGACGTAAGGGATGACGCACTCTAGATGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAAGCAGGAATTCATGAGTAAAGGAGAAG |

## Engineering process

### GFP-XO

GFP-XO is the backbone that was used for cloning of all yeast plasmids. It was ordered from Addgene (Plasmid
#115565).

### pDRF1-GW ymScarletI

pDRF1-GW ymScarletI was the source of the mScarlet gene we used to insert into our yeast constructs. It was
orders from Addgene (Plasmid #118452).

### oxyR-oxyS-GFP

The initial oxyR-oxyS-GFP plasmid was designed in the Panke lab (ETH Zürich) and was kindly provided to us by
Dr. Tsvetan Kardashliev (University of Basel). As it already contained all components for ROS-dependent GFP
expression, this plasmid provided an important baseline for measurements and comparison with further
engineered plasmids.
We first exchanged the promoter region of the original plasmid to modulate oxyR expression, creating the
oxyR-J2311-PL-oxyS-GFP plasmid.
To achieve desensitization of this ROS-reporter to desired thresholds, we employed multiple approaches for
promoter engineering. For the oxyR plasmids, this mainly included error prone PCR and directed mutagenesis on
the oxyS promoter.

### oxyR-J2311-PL-oxyS-GFP

Exchanging the promoter began by excising the old promoter through restriction enzyme digestion with NcoI and
BsphI. The digestion was gel purified and the backbone Gibson assembled with a new\_oxyR DNA fragment from
TWIST. The product (oxyR-J2311-PL-oxyS-GFP) was transformed into DH5ɑ and colonies were checked by sequencing.
TECAN fluorescence assays comparing oxyR-J2311-PL-oxyS-GFP and oxyR-oxyS-GFP indicated that the new promoter
did in fact perform worse than the original. For this reason we decided to focus our engineering efforts on
oxyR-oxyS-GFP.

### Error-prone PCR - random mutagenesis

Error prone PCR is a technique that was utilized to introduce random mutations into the oxyS promoter in
order to influence the binding behavior of oxyR to oxyS. To increase the error rate of Taq polymerase during
PCR amplification, MnCl2 was added in varying concentrations (0.1 - 0.8 mM). The oxyS promoter was then
amplified using the primers old-oxyR-epPCR-fwd and old-oxyR-epPCR-rev. After PCR, DpnI was added to each
reaction to selectively digest the methylated, original DNA. This should reduce the background of unmutated
fragments. The obtained fragments were then cloned into the oxyR-GFP backbone, creating oxyR-epoxyS-GFP
plasmids. Since we had to remove the original promoter from the plasmid to insert the potentially mutated
version, we initially wanted to digest oxyR-oxyS-GFP with restriction enzymes. However, by that time we had
realized that digestions do not work well on that plasmid, and decided to do backbone amplification instead.
We therefore amplified the backbone from oxyR-oxyS-GFP, excluding oxyS, with oldBB-fwd and oldBB-rev primers
and then performed Gibson assembly with the purified epoxyS fragments from epPCR. The products were then
transformed into DH5ɑ and colonies screened by TECAN fluorescence measurements. Promising colonies, namely
oxyR-epoxyS-GFP 0.6 MnCl2 col. 1 and col. 2 were later sequenced. Interestingly, they both did not contain
mutations, despite showing considerably different responses to H2O2.
EpPCR was also attempted for oxyR-J2311-PL-oxyS-GFP. However, we did not manage to generate mutated fragments.

### Directed mutagenesis

After not achieving convincing results with epPCR, we switched to a directed mutagenesis approach. This
happened in tight conjunction with the computational part of our project, as a model was fitted to predict
specific mutations in the promoter sequences that would decrease the binding affinity to the transcription
factor oxyR. A set of 19 DNA fragments containing combinations of these mutations in oxyS were ordered from
IDT. For cloning, we amplified the backbone from oxyR-oxyS-GFP using BB-PCR-GA-F2 and Backbone-PCR-GA\_R
primers and then inserted the dmoxyS\_OG and dmoxyS\_BS fragments via Gibson assembly. DH5ɑ colonies from
transformation were sequenced to confirm cloning success, before being analyzed in TECAN fluorescence assays.
In the end we successfully cloned and tested 5 oxyR-dmoxyS\_OG-GFP and 7 oxyR-dmoxyS\_BS-GFP variants. For the
remaining 7 fragments we did not have enough time left.

### Tga2-CaMV35S-GFP and CaMV35S-GFP

CaMV35S-GFP was constructed by adding tAdh1 and CaMV35S to the GFP-XO backbone. Tga2-CaMV35S-GFP was then
assembled from CaMV35S-GFP by inserting Tga2.
GFP-XO was digested with BamHI and EcoRI.
tAdh1 was amplified from yeast BY4743 genome by PCR using tadh1-FW and CaMV35S-Rv primers, thereby adding
compatible ends for Gibson assembly.
Digested GFP-XO backbone was then assembled with tAdh1 and CaMV35S through Gibson assembly, followed by
transformation into DH5ɑ. Colonies were checked for successful cloning by restriction enzyme digestion of
extracted plasmid. Digestion with EcoRI and SphI was expected to produce fragments of 6845 bp, 626 bp and 331
bp for CaMV35S-GFP. Gel electrophoresis of the digested plasmids revealed expected fragment sizes only for
CaMV35S-GFP colony 4, indicating successful cloning in that colony.
(Figure 5)

![trx2-camv-colonies](https://static.igem.wiki/teams/5356/engineering/an-trx2-camv-colonies.png)

**Figure 5. Trx2-GFP and CaMV35S-GFP gel.**

To add Tga2 to CaMV35S-GFP, the backbone was first digested by BamHI and SpeI. Tga2 DNA fragment
(TGA2\_SpeI\_BamHI) was synthesized by TWIST and inserted into CaMV35S-GFP via Gibson assembly followed by
transformation into DH5ɑ. Colonies were once again checked by digestion and gel electrophoresis of obtained
plasmids. Digestion was performed with SphI and the expected fragments (7337 bp, 1616 bp) were observed for
Tga2-CaMV35S-GFP colonies with varying intensity.
(Figure 6)

![an-trx2-camv-colonies](https://static.igem.wiki/teams/5356/engineering/an-tga2-camv-yap1-colonies.png)

**Figure 6. Trx2-GFP and CaMV35S-GFP gel.**

After receiving the relevant primers, both Tga2-CaMV35S-GFP and CaMV35S-GFP were sequenced, further validating cloning. For testing and quantifying the function of these constructs, they were transformed into yeast BY4741.

### yap1-trx2-GFP and trx2-GFP

trx2-GFP was cloned by adding tAdh1 and Trx2 to the GFP-XO backbone. Similar to the construction of
CaMV35S-GFP, tAdh1 was amplified from yeast genome using tadh1-FW and trx2-Rv primers, the latter making it
compatible for cloning with the Trx2 DNA fragment (UAS\_pTRX2, synthesized by TWIST). tAdh1 and Trx2 were then
inserted into BamHI-EcoRI-digested GFP-XO via Gibson assembly. The product was transformed into DH5ɑ and the
colonies checked by digestion and gel electrophoresis of the obtained plasmids. Digestion with EcoRI and SphI
should produce fragments of 6845 bp, 626 bp and 331 bp length for trx2-GFP, which was observed on the gel,
thereby confirming cloning success (Figure 5).
Later on, the trx2-GFP plasmids were validated by sequencing.
For testing and quantifying the function of trx2-GFP, it was transformed into yeast BY4741.
Once we had successfully cloned the trx2-GFP plasmid, we attempted to add Yap1, the transcription factor that
binds Trx2, to the construct. However, all of the approaches we tried to achieve this, failed.
Initially, we tried restriction enzyme digestion of trx2-GFP with SpeI and BamHI, followed by Gibson assembly
with yap1-Spel and yap1-BamHI. Yap1 was ordered as two halves from TWIST, since it was too large to be
synthesized as one DNA fragment. Transformation into DH5ɑ yielded only one colony which was checked by plasmid
digestion with SphI and gel electrophoresis (Figure 6). The gel revealed that cloning had been unsuccessful.
Digestion and Gibson assembly have been repeated. The Gibson assembly product was amplified by PCR and checked
by gel electrophoresis, indicating issues with assembly or digestion.
We then tried a new Yap1\_BamHI fragment with 21 bp longer (43 bp total) homology to the second fragment to
improve assembly. However, this also failed. After testing different combinations of restriction enzymes with
different conditions and doing gel electrophoresis, we found out that BamHI was not cleaving correctly. Using
a fresh batch of enzyme also did not deliver correct fragments. We subsequently switched to backbone
amplification PCR of trx2-GFP. Unfortunately, we had difficulties amplifying the backbone with correct length
(~7900 bp) and high enough concentration. First, the pTRX2\_BamHI\_F and pTRX2\_SpeI\_R primers were used and
failed. Then, Yap1y-BB-fwd2 and Yap1y-BB-rev primers were designed and tested. With the new primers, we
managed to obtain fragments of correct size, but could not purify it in sufficient amounts. Gibson assembly
with Yap1 DNA fragments did not succeed. We successfully amplified Yap1 from yeast BY4741 genome, yet Gibson
assembly with the backbone did not work. This was likely due to the poor quality of the backbone
amplification.

### Trx2 epPCR

In an effort to reduce affinity of Yap1 to the Trx2 promoter, we performed error prone PCR (epPCR) on the
promoter to introduce mutations. For this we used trx2-epPCR-fwd and trx2-epPCR-rev primers and performed the
reactions at different Mn2+ concentrations ( 0.1 - 0.8 mM) to tune the error rate. Afterwards, the PCR
products were incubated with DpnI to digest the original, non-amplified DNA. The amplified and potentially
mutated Trx2 promoter was inserted into XmaI-EcoRI-digested trx2-GFP (Trx2 removed) and transformed into DH5ɑ.
However, we only obtained non-mutated colonies through this method (determined by sequencing).

### mScarlet plasmids

After measuring strong autofluorescence of yeast during TECAN fluorescence assays, we exchanged GFP to
mScarlet to separate the emission wavelength from that of yeast’s autofluorescence. For this purpose, GFP had
to first be removed from all of the yeast GFP plasmids by restriction enzyme digestion using EcoRI and SalI
and subsequent gel purification. The mScarlet fragment was obtained by digestion of pDRF1-GW ymScarletI with
the same enzymes (gel purification) and then ligated with the backbone. Ligations were transformed into DH5ɑ,
colonies sequenced and correct plasmids transformed into BY4741. TECAN fluorescence assays with mScarlet
plasmids however did not show any signal, regardless of condition. The original pDRF1-GW ymScarletI worked as
expected.
Due to the relative locations of restriction sites on the plasmids, cloning positioned mScarlet relatively far
from the promoter and terminator in our target plasmids (79 bp and 72 bp respectively). We suspected this
could lead to it not being expressed properly. To address this problem, we amplified mScarlet from pDRF1-GW
ymScarletI with mScarletX0-F and mScarletX0-R primers, thereby adding compatible ends for Gibson assembly
closer to the gene itself. The amplified mScarlet was subsequently assembled with EcoRI-digested GFP-XO (GFP
removed) and transformed into DH5ɑ. In the new construct, mScarlet is 49 bp and 27 bp away from the promoter
and terminator respectively. After confirmation by sequencing, mScarlet-XO V2 was transformed into BY4741 and
tested for fluorescence in a TECAN plate reader. This time, mScarlet fluorescence could be detected. Due to
time constraints, we did not manage to reclone, transform, and test all yeast plasmids (trx2-GFP,
Tga2-CaMV35S-GFP, CaMV35S-GFP) with mScarlet V2.

### mScarlet-XO

The mScarlet-XO plasmid was constructed from GFP-XO by exchanging GFP to mScarlet.
First, mScarlet was excised from pDRF1-GW ymScarletI by restriction enzyme digestion with EcoRI and SalI. The
mScarlet fragment was purified from agarose gel after gel electrophoresis. GFP-XO was digested with EcoRI and
SalI to remove GFP. The backbone was gel purified as well.
Digested GFP-XO (GFP removed) was then ligated with the extracted mScarlet fragment using ligase.
Ligations were transformed into DH5ɑ. Emerging colonies were sequenced to confirm cloning.

## Primer sequences

|  |  |  |  |
| --- | --- | --- | --- |
| Primer Name | Sequence (5' - 3') | Location | Use |
| Bacteria Primers | | | |
| Sequencing | | | |
| GFP-fwd-Nter | gcgttcctgtacataaccttcg | beginning of GFP Nter | Promoter sequencing |
| GFP-fwd-Cter | ggcggatttgtcctactcag | just after GFP Cter | Plasmid sequencing |
| Cloning oxyR-J2311-PL-oxyS-GFP | | | |
| Backbone-PCR-GA\_F | catgagggaagcggtgatc | BspHI, SmR gene | BB amplification |
| Backbone-PCR-GA\_R | ggccaacacttgtcactactc | NcoI, in GFP | BB amplification |
| epPCR oxyS promoter | | | |
| old-oxyR-epPCR-fwd | gttcttctcctttgctcatatg | GFP Nter | epPCR |
| old-oxyR-epPCR-rev | Ggtactcaagatcacgaatattc | oxyR Nter | epPCR |
| oldBB-fwd | gaatattcgtgatcttgagtacc | GFP Nter | BB amplification |
| oldBB-rev | tatgagcaaaggagaagaac | oxyR Nter | BB amplification |
| epPCR JS000-PL-oxyS promoter | | | |
| new-oxyR-epPCR-fwd | cagtgaaaagctcttctc | GFP Nter | epPCR |
| new-oxyR-epPCR-rev | caagatcacgaatattcatatcg | oxyR Nter | epPCR |
| BS duplicated version | | | |
| BB-PCR-GA-F2 | cagtgaaaagctcttctc | oxyR Nter | BB amplification |
| Backbone-PCR-GA\_R | see above | GFP Nter | BB amplification |
| Catalase insertion | | | |
| KatG-F | ctcctgctaattaaaaagtaaagtgtgaattcTTACAGCAGGTCGAAACGGTCG | KatG Nter | BB amplification |
| KatG-R | gcattctgactgataattgctcacagcagaattcacactttgtttaactttaagaaggagatttaacatATGAGCACGTCAGACGATATCC | KatG Cter | BB amplification |
| Yeast Primers | | | |
| Yeast backbone | | | |
| Backbone-fwd | ctttacacttctcctatgcac | PacI, beginning of ADH1 promoter | Sequencing |
| TF-fwd | cgtcattgttctcgttccc | end of ADH1 promoter | Sequencing |
| GFP-rev | gggtaagttttccgtatgttgc | GFP Nter | Sequencing |
| Amplifying the Trx2 backbone for yap1 insertion | | | |
| pTRX2\_BamHI\_F | ggatcctaataagcgaatttcttatg | between ADH1 promoter and ter | BB amplification |
| pTRX2\_SpeI\_R | ctagttctagagcggccagc | between ADH1 promoter and ter | BB amplification |
| Yap1y-BB-fwd2 | atcctaataagcgaatttc | between ADH1 promoter and ter | BB amplification |
| Yap1y-BB-rev | actagttctagagcggcc | between ADH1 promoter and ter | BB amplification |
| Adding tADH1 withTrx2 compatibility | | | |
| tadh1-FW | gctggccgctctagaactagtggatcctaataagcgaatttcttatg | tADH1 5' | Genomic amplification |
| trx2-Rv | GATGACAGATCTGAAAGgcccggggggagcgatttgcaggcatttg | tADH1 3' | Genomic amplification |
| Adding tADH1 with CaMV35S compatibility | | | |
| tadh1-FW | see above | tADH1 5' | Genomic amplification |
| CaMV35S-Rv | CCCTTACGTCAGAAGCTgcccggggggagcgatttgcaggcatttg | tADH1 3' | Genomic amplification |
| epPCR for Trx2 promoter region | | | |
| trx2-epPCR-fwd | caaatgcctgcaaatcgctc | XmaI, end of tADH1 | epPCR |
| trx2-epPCR-rev | ctcctttactcatgaattcctgc | EcoRI, end of trx2 | epPCR |
| BB-trx2-epPCR-F | caggaattcatgagtaaaggagaag | EcoRI, end of trx2 | BB amplification |
| BB-trx2-epPCR-R | ggagcgatttgcaggcatttg | XmaI, end of tADH1 | BB amplification |
| mScarlet-X0 V2 | | | |
| mScarletX0-F | ggccgctctagaactagtggatcccccgggctgcaggaattcatgaatggtcatgaatttgaaattg | mScarlet Nter | Genomic amplification |
| mScarletX0-R | catgactcgaggtcgacctgcagcggccatcattatttatacaattcatccattcctccg | mScarlet Cter | Genomic amplification |

{% endblock %}

{% extends "layout.html" %}
{% block tabname %}Contribution{% endblock %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## Protocols

Media

Glycerol cryo-stocks

Add 125µl 100% glycerol to 375µl liquid culture, store at -20° C

Lysogeny Broth

For 1 liter:

* 10g Tryptone
* 5g Yeast extract
* 10g NaCl
* 900ml ddH2O

Fill up to 1 l by adding ddH2O, autoclave.

For selective medium: Add 100 µl of 100ng/µl stock of ampicilin or 50ng/µl stock of streptomycin to 100
ml of LB

For plates: Add 2.5% w/v Agar to medium, heat in microwave until completely liquid, add antibiotic if
needed, pour plates (34
ml/plate needed)

Based on original protocol by Steven Burgess 2016. Lysogeny Broth (LB). protocols.io

YPD

For 1 liter:

* 10g Yeast extract
* 10g peptone
* 900ml ddH2O
* 40mg adenine

After autoclaving, add 100 ml of sterile 20% w/v glucose

For plates: Add 2.5% w/v Agar to medium, heat in microwave until completely liquid, add antibiotic if
needed, pour plates (34 ml/plate needed)

SD-all medium

For 1 liter:

* 1.9g Yeast nitrogen base without amino acids and ammonium sulphate by FORMEDIUM
* 2g Synthetic complete mixture (KAISER), drop-out complete by FORMEDIUM
* 900ml ddH2O

After autoclaving, add 100 ml of sterile 20% w/v glucose

SC-MSG medium

For 1 liter:

* 1.9g Yeast nitrogen base without amino acids and ammonium sulphate by FORMEDIUM
* 1g MSG (L-Glutamic acid monosodium salt)
* 2g Synthetic complete mixture (KAISER), drop-out complete by FORMEDIUM
* 900ml ddH2O

After autoclaving, add 100 ml of sterile 20% w/v glucose

MSG-URA medium

For 1 liter:

* 1.9g Yeast nitrogen base without amino acids and ammonium sulphate by FORMEDIUM
* 1g MSG (L-Glutamic acid monosodium salt)
* 2g Synthetic complete mixture (KAISER), drop-out without uracil by FORMEDIUM
* 900ml ddH2O

After autoclaving, add 100 ml of sterile 20% w/v glucose

For plates: Add 2.5% w/v Agar to medium, heat in microwave until completely liquid, add antibiotic if
needed, pour plates (34 ml/plate needed)

M9 minimal medium

For 1 liter:

* 200ml M9 salts (5x stock solution)
* 1ml 1M MgSO4
* 1ml 0.1M CaCl2
* 20g Glucose
* 900ml ddH2O

Culutre conditions

30° C, shaking for:

* *Saccharomyces cerevisiae* BY4741
* *Saccharomyces cerevisiae* BY4743
* *Pseudomonas putdia* 1320
* *Pseudomonas putdia* 1243
* *Pseudomonas protegens* Pf-5

37° C, shaking for:

* *Escherichia coli* DH5-Alpha
* *Escherichia coli* Nissle 1917

Transformation

Making Heat-shock competent E. coli

#### Materials

##### 1M CaCl2

* 3ml 1M CaCl2
* 100ml ddH2O

Filter sterilize and keep on ice.

##### 0.1M CaCl2

Dilute 1M CaCl2 1:10 in ddH2O.

##### 0.1M CaCl2 + 15% glycerol

* 3ml 1M CaCl2
* 4.5ml sterile glycerol
* 22.5ml ddH2O

Filter sterilize and keep on ice.

#### Protocol

1. Dilute O/N cultures to OD600 0.5
2. Cool culture on ice for 20 min.
3. Centrifuge at 4° C at 4000 rpm for 10 min.
4. Discard supernatant and resuspend in 20ml 0.1M CaCl2.
5. Incubate 30 min on ice.
6. Centrifuge at 4° C at 4000 rpm for 10 min.
7. Discard supernatant and resuspend all pellets in 5ml 0.1M CaCl2 + 15% glycerol.
8. Aliquot 200 µl and store at -80°C (50 µl used per transformation).

Based on original protocol by Chang et al., *JEMI Methods*, Vol. 1:22-25 (2017)

Fast Heat-shock transformation of bacteria

1. Add 1 µl of DNA to 50 µl thawed heat-shock competent cells.
2. Heat-shock for 90 sec at 42°C.
3. Add 100 µl SOC recovery medium from NEB, mix.
4. Streak on a pre-warmed plate, incubate plate at 37°C over night.

Medium Heat-shock transformation of bacteria

1. Add 1 µl of DNA to 50 µl thawed heat-shock competent cells.
2. Incubate for 2 min on ice.
3. Heat-shock 90 sec at 42°C.
4. Incubate for 2 min on ice.
5. Add 100 µl SOC recovery medium from NEB.
6. Incubate at 37°C shaking for 20h.
7. Streak on a pre-warmed plate, incubate plate at 37°C over night.

Long Heat-shock transformation of bacteria

1. Add 2 µl of DNA to 50 µl thawed heat-shock competent cells.
2. Incubate for 30 min on ice.
3. Heat-shock 30 sec at 42°C.
4. Incubate for 2 min on ice.
5. Add 950 µl SOC recovery medium from NEB.
6. Incubate at 37°C shaking for 1h. Preheat plates to 37°C.
7. Streak 200 µl per plate, incubate plate at 37°C over night.

Making Electro-competent E. coli

#### Materials

##### 0.2M MOPS buffer

* 2.093g MOPS free acid
* 0.34g Sodium acetate trihydrate
* 0.146g EDTA
* 40ml ddH2O

Adjust to pH 7 using NaOH, fill up to 50 ml with ddH2O.

Filter sterilize and keep on ice.

##### 0.2M MOPS + 15% glycerol

for 50ml:

* 250 µl 0.2M MOPS buffer
* 15ml sterile glycerol

Fill up to 50 ml with ddH2O and keep on ice.

#### Protocol

1. Dilute O/N cultures to OD600 0.5
2. Centrifuge 2.5 ml at 4° C at 4000 rpm for 10 min.
3. Discard supernatant and resuspend in 10 ml ice-cold ddH2O or 0.2M MOPS + 15% glycerol.
4. Repeat this wash.
5. Centrifuge 1 ml at 4° C at 4000 rpm for 10 min and resuspend in 50 µl ddH2O.
6. Use directly for electroporation.

Based on protocol by Tanaïs Moreau.

Electroporation transformation of bacteria

1. Add 100 ng of plasmid DNA to 50 µl electro-competent cells.
2. Transfer to a 0.1 cm cuvette.
3. Electroporate at 1.8 kV, 200 Ω, 25 µF.
4. Immediately add 200 µl LB.
5. Incubate at 37°C shaking for 2h. Preheat plates to 37°C.
6. Streak 200 µl per plate, incubate plate at 37°C over night.

Yeast comptetency & transformation

#### Materials

##### TE 10x pH 7.5

* 1.21 g Tris Base
* 0.37 g EDTA

dissolve in 100 ml ddH2O and adjust pH to 7.5 with HCl

autoclave

##### LiAc 10x (1M) pH 7.5

* 6.99 g LiAc

dissolve in 100 ml ddH2O and adjust pH to 7.5 with HCl

autoclave

##### PEG4000 50%

* 2.5 g platelets of PEG4000 (PEG3350 also works)
* 1.5 ml ddH2O

mix thoroughly to dissolve and fill up to 5 ml using ddH2O

autoclave

##### sterile DMSO

##### Carrier DNA (10 mg/ml)

Salmon Sperm DNA Solution from Life Technologies/Invitrogen (Product No. 15632-011),
heat denaturation by heating at 100 °C for 5 min and putting on ice immediately

##### Solution A (TE1x, LiAc 0.1M)

* 800 µl sterile ddH2O
* 100 µl TE10x
* 100 µl LiAc10x (1M)

##### Solution B (TE1x, LiAc 0.1M)

* 800 µl PEG4000 50%
* 100 µl TE10x
* 100 µl LiAc10x (1M)

#### Protocol

##### Prepare LiAc competent cells

* Per reaction, add a heaped loop full of cells (2-3 days old colony) to an Eppendorf tube containing 30
  µl of solution A (TE1x, 0.1M LiAc)
* Vortex to resuspend

##### Transformation of LiAc competent cells

1. Add 1-3 µl plasmid DNA [50-100 ng] (the purer the DNA the less you need), mix
2. Do not forget to include a negative control without plasmid DNA and a positive control e.g. an empty
   vector. Latter control allows you to determine your transformation efficiency (see 'Further
   Information')
3. add 5 µl carrier DNA [10 mg/ml], heat denatured, mix.
4. Add 300 µl transformation solution B (TE1x, LiAc 0.1 M, PEG4000 40%),
5. Mix well (vortex).
6. Incubate for 1 h at 30°C.
7. Add 35 µl DMSO, vortex.
8. Heat shock cells for 10 min at 42 °C.
9. Cool down cells immediately on ice for 3 min
10. Spin down cells for 5 min at 800 g in an Eppendorf centrifuge.
11. Remove supernatant by pipetting.
12. Resuspend pellet very carefully in 200 µl SD-all medium.
13. Incubate cells at 30°C for 1 h.
14. Plate cells on selective medium and incubate at 30 °C (first transformants should be visible after 2
    days).

Based on original protocol by Dr. Emmanuel Matabaro form our lab.

DNA extraction/purification

Genomic amplification from yeast

1. Pick one colony of 2-3 days old BY4741 yeast, suspend in 30 µL Zymolase.
2. Incubate at 37°C for 45 min.
3. Amplify fragment by PCR.

* 1 μl DNA (zymolased yeast)
* 1.25 μl forward primer (50 ng/μl)
* 1.25 μl reverse primer (50 ng/μl)

Add ddH2O to reach 12.5 μl, then add 12.5 μl 2x Q5® High-Fidelity DNA Polymerase mix.

Run the following PCR program:

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 7 min |
| 5 cycles | 98 °C | 10 sec |
|  | 59 °C, determined using NEB Tm calculator | 30 sec |
|  | 72 °C | 30 sec/kb |
| 25 cycles | 98 °C | 10 sec |
|  | 72°C | 30 sec |
|  | 72°C | 30 sec/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4 °C |  |

Based on original protocol by NEB Q5® High-Fidelity 2X Master Mix
[NEB](https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492)

General PCR

* 2 μl plasmid
* 1.25 μl forward primer (50 ng/μl)
* 1.25 μl reverse primer (50 ng/μl)

Add ddH2O to reach 12.5 μl, then add 12.5 μl 2x OptiTAQ DNA Polymerase mix.

Run the following PCR program:

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 95 °C | 5 min |
| 25-35 Cycles | 95 °C | 5-10 sec |
|  | 50-68 °C, determined using NEB Tm calculator | 30 sec |
|  | 72 °C | 1 min/kb |
| Final Extension | 72 °C | 7 minutes |
| Hold | 4 °C |  |

Backbone amplification

* 2 μl plasmid
* 1.25 μl forward primer (50 ng/μl)
* 1.25 μl reverse primer (50 ng/μl)

Add ddH2O to reach 12.5 μl, then add 12.5 μl 2x Q5® High-Fidelity DNA Polymerase mix.

Run the following PCR program:

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 sec |
| 25-35 Cycles | 98 °C | 5-10 sec |
|  | 50-72 °C, determined using NEB Tm calculator | 10-30 sec |
|  | 72 °C | 20-30 sec/kb |
| Final Extension | 72 °C | 2 minutes |
| Hold | 4 °C |  |

GeneJET Miniprep by Thermo Fisher

1. Harvest 2ml O/N bacterial culture by centrifugation at 6800 × g in a microcentrifuge for 2 min at room
   temperature. Decant the supernatant and remove all remaining medium.
2. Resuspend the pelleted cells in 250 μl of the Resuspension Solution. Transfer the cell suspension to a
   microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and
   down
   until no cell clumps remain.
3. Add 250 μl of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution
   becomes viscous and slightly clear.
4. Add 350 μl of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6
   times.
5. Centrifuge at 16000 × g for 5 min to pellet cell debris and chromosomal DNA.
6. Transfer the supernatant to GeneJET spin column by decanting or pipetting. Avoid disturbing or
   transferring the white precipitate.
7. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection
   tube.
8. Add 500 μl of the Wash Solution (diluted with ethanol prior to first use) to the GeneJET spin column.
   Centrifuge at 16000 × g for 60 seconds and discard the flow-through. Place the column back into the
   same collection tube. Repeat the wash procedure using 500 μl of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This
   step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube. Add 20 μl of the Elution
    Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact
    the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
11. Measure concentration using Nanodrop and store at -20° C.

Gel electrophoresis

1. Dissolve 1-2% w/v Agarose in TAE buffer by heating in a microwave.
2. Add 0.1% v/v SYBR Safe to the solution & mix.
3. Pour gel into prepared tray.
4. Remove comb inside TAE buffer.
5. Mix PCR sample with 6x loading dye without SDS.
6. Load 5 μl GeneRuler ladder (10 kb) and samples in separate wells.
7. Run electrophoresis at 140 V and 400 mA for 20 min.
8. Inspect gel under UV light.

PCR cleanup using Wizard® SV Gel and PCR Clean-Up System

#### Gel purification:

1. Following electrophoresis, excise DNA band from gel precisely under UV light and transfer to a 1.5 ml
   microcentrifuge tube.
2. Add 10 μl of Membrane Binding Solution per 10 mg of gel slice.
   Vortex and incubate at 50-65 °C until gel slice is completely dissolved.

#### PCR cleanup:

1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly.
   Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000 × g for 1 minute.
   Discard flowthrough and reinsert Minicolumn into Collection Tube.
4. Add 700μl of Membrane Wash Solution (ethanol added).
   Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
5. Repeat wash with 500μl of Membrane Wash Solution.
   Centrifuge at 16,000 × g for 5 minutes.
6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge
   lid
   open to allow evaporation of any residual ethanol.
7. Carefully transfer Minicolumn to a new 1.5 ml microcentrifuge tube.
8. Add 50μl of Nuclease-Free Water to the Minicolumn.
   Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
9. Measure concentration using Nanodrop and store at -20° C.

DNA manipulation

Restriction enzyme digestion

* up to 17 μl plasmid
* 2 μl 10x NEB rCutSmart™ Buffer
* 1 μl of each restriction enzyme

Add ddH2O to reach 20 μl, incubate at 37°C for 60 min.

Gel purify or heat-inactivate restriction enzymes.

Error prone PCR

Work on ice. Higher manganese concentrations introduce more mutations but decrease PCR yield.

* 5 μl 10X ThermoPol Buffer
* 1 μl 10 mM dNTPs
* 2 μl 10 µM epPCR forward primer
* 2 μl 10 µM epPCR reverse primer
* 0.2 μl template DNA
* 0.5 μl Taq DNA polymerase without proofreading activity
* 34.3 μl ddH2O
* 5 μl 0.1-0.8 mM MnCl2

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 95 °C | 60 sec |
| 30 Cycles | 95 °C | 30 sec |
|  | 54 °C , determined using NEB Tm calculator | 30 sec |
|  | 68 °C | 60 sec |
| Final Extension | 68 °C | 2 minutes |
| Hold | 4 °C |  |

Gel purify PCR product and gibson assemble into backbone.

Optional: Add 1 μl DpnI enzyme to digest methylated DNA (background)

Gibson assembly

Work on ice.

* 1 μl (50-100ng) digested backbone DNA
* 2 μl (2-3x excess) of each DNA fragment

Add ddH2O to reach 5 μl, then add 5 μl 2x Gibson Assembly® Master Mix by NEB.

Incubate at 50°C for 1h, then transform or store at -20°C.

Ligation

Work on ice.

* 1 μl (50 ng) digested backbone DNA
* 2 μl (37.5 ng) of each DNA fragment
* 1 μl 10x T4 DNA Ligase Buffer by NEB

Add ddH2O to reach 9 μl, then add 1 μl T4 DNA ligase by NEB.

Incubate at RT for 15 min or 1 h, then transform or store at -20°C.

Fluorescence Measurement

Fluorometric 96 well plate assay

1. Calculate volume of overnight culture needed for dilution to OD600=1 in 3.5 ml final
   volume.
2. Transfer calculated amount into a falcon tube.
3. Centrifuge at 4500 x g for 5 min and resuspend in 3.5 ml PBS.
4. Dilute H2O2 stock solution (10 M) to the following concentrations: 0.01 mM, 0.1
   mM, 1 mM, 2 mM, 5 mM,
   10 mM.
5. Prepare a clear bottom black walls 96 well plate (Greiner 655097) and add 180 μl of resuspended cells
   + 20 μl H2O2 H2O2
   per well. The final concentration in the well will be 10x lower than that of the prediluted
   H2O2 solution.
6. Load plate into TECAN plate reader and start the following program.

TECAN plate reader protocols

#### Yeast OD600 + GFP

1. Heat to incubating temperature (30° C), shake for 30 sec.
2. Every 5 min, shake for 10 sec, wait for 2 sec.
3. Measure OD600 and fluorescence, using 10 flashes per well, measure at bottom.
   For GFP use Ex λ: 495 and Em λ: 530; Gain 140;

#### Yeast OD600 + GFP + mScarlet

1. Heat to incubating temperature (30° C), shake for 30 sec.
2. Every 5 min, shake for 10 sec, wait for 2 sec.
3. Measure OD600 and fluorescence, using 10 flashes per well, measure at bottom.
   For GFP use Ex λ: 495 and Em λ: 530; Gain 140;
   for mScarlet use Ex λ: 569 and Em λ: 594; Gain 50;

#### Bacterial OD600 + GFP

1. Heat to incubating temperature (37° C), shake for 30 sec.
2. Every 5 min, shake for 10 sec, wait for 2 sec.
3. Measure OD600 and fluorescence, using 10 flashes per well, measure at bottom.
   For GFP use Ex λ: 495 and Em λ: 530; Gain 140;

Fluorometric ROS detection Kit by Sigma-Aldrich

Before first use, add 40 μl of DMSO to the vial of DCFDA and mix well. Store at -20°C in the dark.

1. Prepare Reaction Master Mix: To 1 ml Assay buffer, add 2 μL ROS + DMSO detection reagent stock
   solution.
2. Centrifuge the cells from overnight culture and resuspend the cell pellets in PBS to reach OD 0.001 in
   90 μL (at 100,000 cells/well) in a 96-well plate.
3. Add 110 μL of H2O2 solution to the cells (Final concentrations: 0, 0.1, 1 mM).
4. Incubate and measure in TECAN for 1h min at 37°C (Ex 520, Em 605).
5. Centrifuge the plate at 2250 x g for 7 minutes with brake off.
6. In half the wells, remove supernatant and replace with assay medium and resuspend, resuspend other
   pellets.
7. Incubate in TECAN for 1h min at 37°C.
8. Measure fluorescence in TECAN (Ex 520, Em 605).
9. Centrifuge the plate at 2250 x g for 7 minutes with brake off.
10. In half the wells, remove supernatant and replace with PBS and resuspend, resuspend other pellets.
11. Incubate in TECAN for 2h min at 37°C.
12. Centrifuge the plate at 2250 x g for 7 minutes with brake off.
13. Remove supernatant and replace with assay medium and resuspend and measure fluorescence in TECAN (Ex
    520, Em 605).

{% endblock %}

{% extends "layout.html" %}
{% block tabname %}
Results
{% endblock %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Did we manage to desensitize our ROS sensor? In bacteria and yeast? What were our discoveries?

Initial ROS-sensing assessment

Bypassing OxyR downregulation

Random mutagenesis

Directed mutagenesis

Host-induced variability

Testing the catalase output

Yeast version of our sensor

Conclusion & outlook

## Initial oxyR-oxyS-gfp system sensitivity assessment

In the **oxyR-oxyS-gfp plasmid**, the oxyRS regulon is activated by the oxidized form of the oxyR transcription factor, inducing GFP reporter expression and oxyR downregulation. After transforming the oxyR-oxyS-gfp in *E. coli* DH5a, fluorometric assays were performed. The GFP fluorescence signal was aquired over several hours, at t=0 H2O2 was added in different concentrations (TECAN Infinite® M Nano+ 96-well plate reader). For all further graphs, **RFU (Relative Fluorescent Unit)** is calculated as fluorescence normalized by OD600, and **RRFU** is the relative RFU compared to the 0 mM H2O2 RFU.

An increase in GFP expression was measured after addition
of H2O2, for all concentrations (Figure 1). The intensity of the GFP signal correlates positively
with the
concentration
of H2O2, a higher concentration inducing a stronger signal. An initial exponential
increase is observed, where the oxidative stress is strong,
followed by a linear weaker increase after 1 hour, translating the stabilizaton of the cell system. As expected, the bacteria inoculated with no
H2O2 only
showed a baseline fluorescence and no increase over time.

This shows our **reporter system is relevant in monitoring H2O2-induced oxidative stress** in bacteria over time.
Here, we see that even low H2O2 concentrations induce high GFP signals, expected from **ultra-sensitive native** oxidative-stress responsive systems, which confirms the need to **desensitize** our oxyR-based sensing system.

![Example of how our engineered microbiota could serve as an infection detector and biocontrol agent.](https://static.igem.wiki/teams/5356/results/20240821test-oxyr-dh5a.png)

**Figure 1. oxyR-oxyS-gfp in *E. coli* is too sensitive.** RFU over time for various starting concentrations of H2O2. GFP fluorescence (at λ=530 nm) measurement of oxyR-oxyS-gfp transformed *E. coli* DH5a, normalized for OD600.

![Barchart.](https://static.igem.wiki/teams/5356/results/20240917-fluorescence-barchart-oxyr-dh5a.png)

**Figure 2.**  Corresponding RRFU representation to Figure 1. for
timepoints at 15min, 1h, 2h and 3h. The largest increase in fluorescence occurs in the first hour, and the stongest step between H2O2 concentrations of 1 mM and 5 mM.

To assess robustness of the system, we tested a second stimulation after 2h to see if bacteria was still susceptible to oxidative stress or if **durable tolerance** was to be forseeable.

Visible in Figure 3, late H2O2 addition (+2h) leads to a second increase in fluorescence, albeit not as steep as the initial peak. The curve flattens out as intracellular H2O2 is depleted
and the oxyS promoter
does not express much GFP anymore. GFP is degraded slowly, but is probably constantly produced due to
oxidative stress from nutrient deprivation in PBS.

![Addition after 2h.](https://static.igem.wiki/teams/5356/results/2024-09-25-oxyr-2mm-2haddition.png)

**Figure 3. oxyR-oxyS-gfp system could be used durably over time.** RFU over time for *E. coli* treated with 2mM H2O2. After 2 hours, H2O2 was added at a final concentration of 10 mM to all samples marked with 2h. Two colonies of non-transformed and transformed DH5a were tested in each condition.

## Placing a iGEM-tested constitutive promoter in front of OxyR, thereby stopping the negative feedback-loop

To obtain a **stable signal** of infection, we want a **stable expression** of our output protein. To **bypass oxyR’s negative impact on its regulation**, we redesigned the promoter region to include a **constitutive promoter** for oxyR (See  [Design & Engineering](engineering) page). After successful transformation of the constructed
**oxyR-J23101-PL-oxyS-gfp "new oxyR"** plasmid into *E. coli* Nissle (confirmed by sequencing), we compared it to the
oxyR-oxyS-gfp in *E. coli Nissle*. We used Nissle as this strain was the **most relevant recipient** for our human health application.

![New oxyR.](https://static.igem.wiki/teams/5356/results/20240821-new-oxyr.png)

**Figure 4. The "new oxyR" plasmid does not show any activity.** RFU over time for oxyR-J23101-PL-oxyS-gfp VS oxyR-oxyS-gfp transformed *E. coli* Nissle at different starting H2O2 concentrations.

As seen in Figure 4, the new oxyR plasmid did not show any GFP
expression,
being indistinguishable from Nissle without a plasmid. This may be due to **loss of plasmid** between sequencing
and TECAN measurement (storage at -20°C) or an **issue in the plasmid design**, leading to no GFP expression. The baseline GFP fluorescence at 0 mM H2O2 can be attributed to oxyS promoter leakiness.
Finally, we can notice the reduced fluorescence for the 10 mM H2O2
concentration compared to 5 mM, which is
most
likely due to **cell death under high oxidative stress**. In practice, we would hope for the population to be fit enough to **withstand** such concentrations, as many bacterial microbiota already do by **tolerance and adaptation**.

## Random mutagenesis of the oxyR-oxyS-gfp plasmid

To raise the ROS concentration threshold needed for GFP expression, as we need a **high-pass ROS filter**, we aimed to mutate the promoter region by error-prone PCR (**epPCR**, see  [Design & Engineering](engineering) page). Many colonies were picked from each epPCR and characterized. Figure 5 shows the reduced GFP expression of colony 2 from the 0.6 mM MnCl2 epPCR. Results show overall decreased response but no change in concentration-specific behaviours. Sequencing revealed that error-prone-PCR did **not yield any mutations** in the picked colonies. We hypothesize that **stress** from Gibson
Assembly and transformation led to transcriptional changes impacting ROS-sensitivity and our sensor system.

![Barchart.](https://static.igem.wiki/teams/5356/results/240922-errorbar-0-6-col2.png)

**Figure 5. epPCR was not successful, but some colonies had interesting ROS responsiveness.**  RFU over time of colony 2 (0.6 mM MnCl2 epPCR).

Further colonies were picked from the epPCR plate and measured, again without actual sequence mutations, and also demonstrating different ROS-response profiles as shown in Figure 6. Even though the Taq polymerase used has no
proofreading ability and its error-rate is increased by high manganese concentration, **its error-rate may still
be too small for the short targeted promoter sequence**, leading to no mutations.

The variation in phenotype being small and not high-pass -like, **we chose to put random mutagenesis to the side**, as we did not manage to generate mutants.

![Barchart.](https://static.igem.wiki/teams/5356/results/20240919-eppcr-fluorescence-barchart-combined.png)

**Figure 6. A more complete RRFU analysis of 3 epPCR colonies showing modified ROS-responsiveness.** RRFU over time of colony B, C and 2 (0.6 mM MnCl2 epPCR).

## Directed mutagenesis of the oxyR-oxyS-gfp plasmid

In order to tune our system to be a **high-pass filter of ROS**, i.e. getting activated from 1mM H202 (ideally 5mM for plant infections) while conserving high output expression, we turned to **computational modeling** to determine
relevant targeted point-mutations which would make the system better fit these criterions. The model was based on local decrease of transcription factor (OxyR) binding affinity to the promoter. We ran our model on both designed ROS-sensing systems, e.g. the **oxyR-dmoxyS\_OG-gfp** and the **oxyR-dmoxyS\_BS-gfp** (with a **duplicated binding region(=BR)**)
An array of mutation combinations for both promoter versions (BR duplicated or not) was designed, cloned and transformed into DH5-alpha. OG and BS mutations respectively (8 mutated versions each) were predicted to have slight to strong impact on OxyR binding affinity to its promoter (See corresponding[Design section](engineering.html#section3) and [Model page](model) for details.)

In Figure 7, we represented the most relevant constructs, which show significantly different ROS-responsiveness characteristics. For instance, OG-4 shows improved GFP expression at 10 mM H202 compared to the unmutated control, but the best improved construct would, in fact, be OG-3 since the response is still high at high concentrations, but the background is more than twice as low for low concentrations, which is what we were looking for in our high-pass filter designs. As expected, versions with too many mutations showed impaired ROS-responsiveness, as seen for OG-1 (4 soft mutations). These results also show the importance of **experimental support and verification of computational models**, as predicted soft mutations did not induce the same impact on ROS-sensing, as seen comparing OG-2, OG-3, OG-4 and OG-5 (all with a single soft mutation).
Here, we also show the **success of the design and engineering of our BR-duplicated version** , oxyR-dmoxyS\_BS-gfp, where **BS-4** more specifically shows promising high-pass filter characteristics.

![OG, BS](https://static.igem.wiki/teams/5356/results/20240917-fluorescence-barchart-combined.png)

**Figure 7. Improved ROS-sensing constructs were generated through directed mutagenesis.** RRFU over time for all relevant mutants compared to the unmutated control.

In order to better analyze our data and for it to be **relevant to our end application** perspectives, we represented the **ratios of RFU for 5 mM compared to 1 mM H202**. Indeed, for plant infection detection, we would need our system to **only be activated from 5 mM** (ROS levels observed during fireblight infection) and **inactive around 1 mM** (baseline variations expected due to other abiotic stresses). Here, we confirm OG-3 being the best improved version with a 40% increase in fluorescence (Figure 8). Further experiments should be done for OG-2, since we have observed sky-rocketting fluorescence and, for this experiment, had to dilute it 1:10 so the signal could be properly detected without saturation.

![OG, BS](https://static.igem.wiki/teams/5356/results/fluo-ratio-og-bs-total-5mm-to-1mm-2h-1.png)

**Figure 8. Corresponding Ratio plot showing that OG-3 is the best improved mutant for high-pass ROS sensing.** RFU Ratio of 5 mM to 1 mM H202 (high-pass relevant for the plant infection detection application) for the same experiment as Figure 7.

## Assessing host-induced variability of ROS-sensing

To adapt our **ROS sensor to our different applications**, our system had to show high-pass ROS-filter -relevant characteristics in our **end application host strains**, e.g. *E.coli* Nissle for IBD treatment and yeast or plant bacterial strains for fireblight detection. In the results shown Figure 9, we see how the **choice of host organism** is crucial to take into account as it **highly impacts the ROS-responsiveness profiles**. Most interestingly, *E.coli* K-12 and Nissle show decreased GFP-induction at lower concentrations while keeping high expression at high concentrations (from 2 mM for K-12 and 5 mM for Nissle) compared to DH5a, which is **in favour of our high-pass filter objective**. However, in humans, 5 mM H2O2 is not typically reached, and this raises the question of the pertinence of **Nissle chassis for ROS-sensing in humans**, as it would need further adjustements to rather respond **strongly from 1 mM**. Finally, we also tested **high-ROS tolerant** strain like the *Pseudomonas alloputida* mt-2 KT244 (1320), as we thought we might observe tolerance at lower concentrations and response only at high ROS concentrations (**high-pass**), but the 1320 strain seemed to be **broadly tolerant to oxidative stress** and, thus, not react at all. The **1243** strain does show **high-pass behaviour**, but the signal obtained at high-ROS could be **too low for drone detection**.

![OG, BS](https://static.igem.wiki/teams/5356/results/20240905-hosts-fluorescence-barchart-combined.png)

**Figure 9. High impact of host choice in ROS-sensing behaviour.** RRFU over time. The oxyR-oxyS-gfp plasmid was transformed in 5 different bacteria strains. *P.putida*
strains were not included for 2 mM H2O2 concentration due to experimental choices.

## Testing the catalase output in oxyR-oxyS-gfp system (IBD model)

We are aiming to develop a **theragnostic** system, which associates **high ROS detection** to **local production** of anti-oxidant enzymes to **counteract the ROS imbalance**. Indeed, in our chosen human disease case-study, IBD, ROS are at the center of a **perpetual positive feedback loop**, where inflammation triggers more ROS which triggers more inflammation.

Therefore, we designed a construct with the **anti-oxidant enzyme catalase** as a supplementary therapeutic output to the GFP reporter, called **oxyR-oxyS-KatG-gfp** (See the  [Design & Engineering](engineering) page for more details). We chose **not** to express the catalase **constitutively** but link it to **ROS sensing** in order to preserve bacteria **fitness**, mimicking native ROS pathways which produce anti-oxidant enzymes only when needed.

![Catalase.](https://static.igem.wiki/teams/5356/results/catalase-comp.png)

**Figure 10. Catalase induction serves to maintain H2O2-induced oxidative stress low.** RRFU analysis of oxyR-oxyS-gfp and OxyR-OxyS-KatG-gfp transformed bacterial strains for different H2O2 concentrations.

Catalase converts H2O2 into dioxygen and water. To assess its **effectiveness in degrading ROS** in our model assay, we tested to different quantifications, one **direct** through intracellular ROS titration, and the other **indirect** through RFU monitoring of our reporter GFP. Sadly, we did not manage to make the intracellular ROS-titration kit work in our timeframe, so we will only show our RFU data.
In Figure 10, we observe **significantly lower** fluorescence levels and increase for the KatG plasmid version compared to the control oxyR-oxyS-gfp. Overall lower expression levels were expected since 2 genes are now under the same promoter, but the slower increase also suggests less activation of the promoter, so less oxidative stress and thus elimination of H2O2 molecules. Our interpretation is that  **catalase expression is successfully induced** and participates in regulating the H2O2 levels, thereby **keeping oxidative stress low**, in turn leading to less reporter GFP expression.
These results show the promise of our theragnostic platform for IBD treatment, although more direct titrations are necessary to be able to validate the hypothesis above.

## Our sensor tested in yeast

### Cloning the Trx2-gfp constructs

For the plant application, we developped in parallel of our bacterial platform, our yeast platform, since **yeast have shown to be effective as biocontrol agents**. Instead of the bacterial ROS-sensing TF OxyR, we used the **native yeast TF YAP1** and the **plant TF TGA2** (expressed in yeast), designed as **yap1-trx2-gfp** and **Tga2-CaMV35S-gfp** constructs (See  [Design & Engineering](engineering) page). Here, we show **only YAP1 data**, as TGA2 did not show any response and we think **co-activators** present in the plant should have been **co-expressed** in yeast for the system to work.

![TRX2.](https://static.igem.wiki/teams/5356/results/240911-trx2-gfp.png)

**Figure 11. Switch-like behaviour of the trx2-gfp sensor in *S. cerevisiae*: a promising high-pass that needs further tuning.** GFP fluorescence of TRX2 depending on H2O2 levels over time.
GFP expression is low
under a threshold, and is much stronger at concentrations 1 mM H2O2 and above.

As a reminder, trx2 is the promoter region bound by the oxidative-stress regulated YAP1 TF
naturally present in *S. cerevisiae*. We did clone the **trx2-gfp** plasmid but did not manage to insert the yap1 gene. Since **YAP1 is natively expressed**, we could still test the trx2-gfp construct.
The trx2-gfp showed nearly a **3-fold increase** in RFU from 1 mM H2O2 and background fluorescence below, which would be perfect for the human application but, for the plant application, we would need activation **only from 5 mM H2O2**. These results are **very encouraging**, trx2-gfp exhibiting a switch-like behaviour. Further, we would also do **directed mutagenesis** on the **trx2 promoter** in order to **shift the activation threshold from 1 mM to 5 mM H2O2** and obtain our ideal high-pass filter.

### Solving spectra overlap issues

GFP exhibits an emission wavelength which overlaps too much with the **autofluorescence of yeast and plants**, so a reporter gene was selected to replace GFP, **ymScarlet**, which would be compatible with observed autofluorescences.

After our first cloning (digestion and ligation), we observed no ymScarlet emission (λ=594) for **mScarlet-X0 "V1"**, as shown Figure 12. This probably results from design of the ligation,
where the 5' and 3' distances after the promoter and before the terminator were changed slightly, thus leading in **expression hindrance**.

We tried again through Gibson Assembly (amplifying the backbone). This time, **mScarlet-X0 "V2"** showed **very high** ymScarlet signal, demonstrating the **success** of our design and engineering, and our **DESIGN-BUILD-TEST** cycle approach.

Due to time restrictions, this process was not yet repeated for all other
constructs.

![Catalase.](https://static.igem.wiki/teams/5356/results/mscar-xo.png)

**Figure 12. Successful DESIGN-BUILD-TEST cycling to obtain ymScarlet expression in *S.cerevisiae*** ymScarlet fluorescence (λ=594) of ymScarlet-X0 V2 compared to ymScarlet-X0 V1 construct.

## Conclusion & outlook

We have successfully engineered mutant variants of the OxyR-based sensor system which show improved high-pass sensor characteristics (see OG-3 Figure 8). Further tuning and characterizations would allow for more knowledgeable selection of a good candidate to pursue the process of developping the theragnostic system with. Indeed, further titrations are needed to properly validate the effectiveness and mode of action of the catalase output (Figure 10), and should be put under the control of the selected improved oxyR promoter. Further down the line, for biosecurity reasons and stability purposes, the sensing system should be genomically integrated in the bacterial/yeast DNA. Genomic integration will surely impact ROS-responsiveness and the system will have to be carefully re-characterized. For IBD treatment, a kill-switch should also be integrated.

We noticed non-replicability of assays using the same colonies from the same cryostocks, where cells seemed to lose ROS-responsiveness and we think lost their plasmid during storage, since the stocks were mistakenly stored at -20°C rather than -80°C. This left us unable to repeat
some characterizations under standardized conditions.
We would retransform some of the plasmids to generate more reliable and
standardized data, especially for the yeast constructs.

In yeast, we obtained very promising results for the trx2-gfp construct, exhibiting a switch-like behaviour, with an distinct activation from 1 mM H202 (Figure 11). In order to shift the activation from 1 mM to 5 mM H202 (relevant range in plants), we would do directed mutagenesis on the trx2 promoter, in the same way as for the oxyR-based sensor.
Ideally, in yeast, for better measurments and end-application compatibility, we would also switch all GFPs to ymScarlet, avoiding overlaps with natural yeast and plant autofluorescence ranges.

Future plans include transforming Pf-5 bacterial biocontrol strain for the plant application, as an alternative to yeast.

All constructs would be tested in a plant model in vitro. We had planned to co-inoculate a cut flower blossom with the pathogenic and engineered sensor bacteria/yeast at Agroscope, but did not have suitable candidates to test yet.

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