Team:ETH Zurich/Notebook

PAVLOV'S COLI

NOTEBOOK

JULY

WEEK 1 (27.6. – 5.7.)

Test 1A: Construction of pNorV and norR plasmids

We ordered gBlocks for:

  • norR without forbidden restriction sites
  • two versions of pNorV: one with the native spacer after transcription start site and one without

Test 1B: Construction of promoters with esaboxes and esaR plasmid

We ordered gBlocks for promoters with esaboxes. E. coli colonies with plasmid with esaR from addgene arrived.

Test 3: Switch based on recombinases

We ordered gBlocks for 3 different codon optimized recombinases without forbidden restriction sites:

  • bxb1
  • phiC31 and
  • tp901

General

  • We ordered first oligos
  • We prepared first TFB1 and TFB2 buffers for competent cells. The next day we prepared the first batch of competent TOP10 cells (80 transformations).
  • We did first transformations:
    • interlab study plasmids
    • pSEVA backbone plasmids
    • plasmids from distribution kit to get J23118 promoter, terminator, prefix and suffix
    • Transformation of plasmids with fluorescent proteins we might use: sfGFP, mCherry, mNectarine, mTurqouise.
  • Followed were first overnight cultures of transformations and first minipreps of the plasmids from transformations and addgene colonies.
  • We prepared first batch of ingredients for M9 media.
  • We poured first LB-agar plates with single resistances for carbenicillin, kanamycin and chloramphenicol.

WEEK 2 (6.7. – 12.7.)

Test 1A: Construction of pNorV and norR plasmids

We used two approaches to get norR and pNorV fragments:

  • PCR to extract genomic copy of norR and pNorV
  • PCR to multiply norR and pNorV from gblocks.
Comment: Genomic PCR was not successful because we added too much bacterial culture. It was not repeated after we successfully multiplied norR from gBlock.

Test 1B: Construction of promoters with esaboxes and esaR plasmid

We did PCR to get a fragment with esaR from addgene plasmid and added overhangs to it.

Test 3: switch based on recombinases

We did site directed mutagenesis (PCR) to add ssRa tag to mNectarine and sfGFP.

Directed evolution: make EsaR specific to AHL present in the gut

  • We prepared electro-competent DH5alpha -uptr cells.
  • We transformed respective cells with plasmid with uracil phosphoribosyltransferase (upp or uptr).
  • - We performed Tecan plate reader experiment to test the response of cells with and without the plasmid towards 5-fluorouracil. Upp/gfp plasmid is under control of dmpR/phenol.
    • 5-FU had an inhibitory effect on growth with all concentrations. There was no growth difference between un-induced and induced state
  • We attempted to construct CAT-UPTR fusion protein:
    • We did PCR to get fragments with CAT (chloramphenicol resitance) and UPTR.
    • We did Gibson asesembly of CAT and UPTR fragments to create fusion protein.
  • We attempted to construct hsvTK (herpes simplex virus thimidine kinase)-APH-Stop-GFP operon:
    • We did PCR to create APH and hsvTK fragments and performed Gibson assembly. Colony PCR did not show any results. However, since the cells grew, they were Amp resistant.

General

  • We repeated failed transformations.
  • We transformed additional pSEVA empty backbone plasmids.
  • We did PCR to linearize pSEVA backbones and to get fragments with prefix + J23118 and terminator + suffix for Gibson assemblies.
  • We did digestion to exchange oris on two plasmid backbones.
  • Following all successful transformations we did overnight culture and minipreps.

WEEK 3 (13.7. – 19.7.)

Test 1A: Construction of pNorV and norR plasmids

We performed a Gibson assembly to create a norR plasmid with J23118 promoter and T0 terminator.

Test 3: Switch based on recombinases

  • We did PCR to create fragments with overhangs for Gibson assembly for parts containing recombinases phiC3, bxb1 and tp901, with attB site in front of recombinase and attP site after terminator.
  • We did transformations of sfGFP + ssRa and mNectarine + ssRa plasmids created a week before. Adding ssRa tag to sfGFP was not successful.

Directed evolution

We did PCR of fragments for construction of hsvTK-GFP with overlaps to esaboxes.

General

  • We repeted all previously failed digestions to exchange resistances on empty backbone plasmids.
  • After transformations of plasmids created with GA or site directed mutagenesis, colonies were picked in the evening for overnight culture and miniprep was done the next day in the morning.

WEEK 4 (20.7. – 26.7.)

Test 1A: Construction of pNorV and norR plasmids

  • We did PCR to construct promoter library for norR
  • We did PCR to add overlaps to norR fragment and repeat Gibson assemblt for norR
  • We did Gibson assembly of pNorV + sfGFP plasmid. Followed we did PCR to create fragment with insert pNorV+sfGFP to put it in a different backbone.

Test 1B: Construction of promoters with esaboxes and esaR plasmids

  • We multiplied with PCR fragmets from gBlocks of promoters with esaboxes.
  • We created fragment with RBS+EsaR from addgene plasmid
Comment: We had problem with constructing J23118+esabox plasmids: restriction did not yield the correct fragment size.

Test 3: Switch based on recombinases

  • We did site directed mutagenesis (PCR) to add moderate ssRa tag to mNectarine.
  • - We did PCR to create remaining fragments for Gibson assembly of our initial design for reporters for recombinases:
    • fragments containing bxb1, tp901, phiC31
    • terminator
    • mNectarine
    • tetR
Comment: At the end of the week all fragments for the reporter were created

WEEK 5 (27.7. – 2.8.)

Test 1B: Construction of promoters with esaboxes and esaR plasmids

  • We did PCR of fragments to construct esaR plasmid:
    • empty backbone from norR plasmid to put esaR in place of norR
    • fragment with overlaps and RBS+EsaR
  • We performed Gibson assembly to create esaR plasmid.

Test 2: AND gate promoter pNorV+esabox

We did restriction and ligation to create norR + esaR plasmid on medium copy plasmid

Test 3: Switch based on recombinases

  • We did PCR to create fragment with RBS+TetR with overhangs and Gibson assembly to create tetR plasmid
  • We performed Gibson assembly of plasmid with mNectarine.
  • We did PCR to generate fragments with the according attB site in front of recombinases bxb1, phiC31 and tp901 and fragments with according attP sites after the terminator
Comment: at the end of the week all fragments for the assembly of reporters are ready, except sfGFP with ssRa tag.

General

  • Ori exchange in plasmid did not work with restriction, we tried a new approach with two part Gibson assembly.
  • We were experiencing problem with gel cleanup, which often yielded impure fragments. Impurities from the fragment might inhibit Gibson assembly reactions. We tested gel extractions with different elution buffers: water, elution buffer from the kit and Tris-HCl. We decide to use Tris-HCl.
  • We 3D printed a stand with magnetic beads for purification of gel extractions with magnetic beads

AUGUST

WEEK 6 (3.8. – 9.8.)

Test 1A: Construction of pNorV and norR plasmids

  • We repeated Gibson assembly to create two versions of pNorV + sfGFP plasmids.
  • We did an ori exchange to create low copy plasmid with norR.

Test 3: Switch based on recombinases

Results from model simulations show that the design where recombinase is not embedded between the recombinase recognition sites is better. We changed the original design into a three plasmid system and we redesigned plasmids and order oligos.

General

We prepared competent cells (160 transformations).

WEEK 7 (10.8. – 16.8.)

Test 1A: Construction of pNorV and norR plasmids

  • We did colony PCR and sent to sequencing pNorV+sfGFP plasmids
  • We did site directed mutagenesis to add moderate ssRa tag to sfGFP in a plasmid with pNorV+sfGFP. Sequencing results showed a missing terminator and ssRa tag. Addition of ssRa tag was not successful.
  • We did first double transformations of pNorV+sfGFP and norR plasmids with additional double transformations of pNorV + empty backbone as a control.
  • We did a plate reader experiment. Results were inconclusive due to dilution error. As a source of nitric oxide (NO) we used DETANO.

Test 1B: Construction of promoters with esaboxes and esaR plasmids

We performed restriction and ligation to create a low copy plasmid version of esaR plasmid.

Test 3: Switch based on recombinases

  • We received a plasmid with bxb1 recombinase from Bonnet lab.
  • We transform plasmid from the distribution kit with ptet promoter.
  • We did resistance exchange on pSEVA291 backbone.
  • We did PCR to create fragments without attP and attB sites next to recombinases and performed Gibson assembly of the new design for the recombinases.
  • We created a low copy plasmid version of tetR.

General

We prepared competent cells and poured plates with double and triple resistances. We 3D print rack for sorting of white tips.

WEEK 8 (17.8. – 23.8.)

Test 1A: Construction of pNorV and norR plasmids

  • We repeated addition of ssRa tag to pNorV+sfGFP with site directed mutagenesis.
  • Sequencing revealed deletion in norR gene.
  • We performed plate reader with low copy number plasmid versions of pNorV+sfGFP and norR plasmids:
    • In the plate reader experiments we did a time and dose response curve for strains with pNorV and norR plasmid and with pNorV and empty backbone. Concentrations of NO from DETANO were based on a model developed by our modelers. Due to small amount of DETANO remaining in the lab we were only able to use concentrations in the lower range of the dose response. The difference between the fluorescence of positive control and pNorV plasmid strains additionally supports our assumption. Most importantly, get a dose response even when norR plasmid is not present in the cell. Results from this experiment show we get an induction of pNorV when the DETANO is present in the medium with or without norR plasmid.
    Based on the plate reader experiment and on sequencing results which show deletion in norR we concluded the NorR from our plasmid is not responsible for the activation of transcription and the dose response we observe. We made a hypothesis that NorR from a genomic copy of norR is binding to the pNorV promoter on our plasmid.

Test 1B: Construction of promoters with esaboxes and esaR plasmids

We created a plasmid with KpnI site between J23118 promoter and sfGFP to enable addition of esaboxes with restriction and ligation. After that, we added esaboxes behind J23118 promoter using restriction and ligation.

Test 3: Switch based on recombinases

  • Colony PCR of Gibson assembly from the previous week did not show any positive results. We repeated Gibson assembly for plasmids with recombinases under tet promoter.
  • We put mNectarine on a medium copy plasmid

WEEK 9 (24.8. – 30.8.)

Test 2: AND gate promoter pNorV+esabox

  • We did Gibson assembly of plasmid with esaR and norR on a low copy plasmid.
  • We performed a restriction and ligation to create hybrid promoters for the AND gate: We created 10 different combinations of promoters:
    Hybrid promoter
    pNorV_native_spacer_esabox
    pNorV_native_spacer_esabox_8bp_spacer_esabox
    pNorV_native_spacer_esabox_8bp_spacer_esabox_8bp_spacer_esabox
    pNorV_native_spacer_esabox_15bp_spacer_esabox
    pNorV_native_spacer_esabox_15bp_spacer_esabox_15bp_spacer_esabox
    pNorV_no_spacer_esabox
    pNorV_no_spacer_esabox_8bp_spacer_esabox
    pNorV_no_spacer_esabox_8bp_spacer_esabox_8bp_spacer_esabox
    pNorV_no_spacer_esabox_15bp_spacer_esabox
    pNorV_no_spacer_esabox_15bp_spacer_esabox_15bp_spacer_esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

SEPTEMBER

WEEK 10 (31.8. – 6.9.)

Test 1A: Construction of pNorV and norR plasmids

Test 1B: Construction of promoters with esaboxes and esaR plasmids

Test 2: AND gate promoter pNorV+esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

WEEK 11 (7.9. – 13.9.)

Test 1A: Construction of pNorV and norR plasmids

Test 1B: Construction of promoters with esaboxes and esaR plasmids

Test 2: AND gate promoter pNorV+esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

WEEK 12 (14.9. – 20.9.)

Test 1A: Construction of pNorV and norR plasmids

Test 1B: Construction of promoters with esaboxes and esaR plasmids

Test 2: AND gate promoter pNorV+esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

WEEK 13 (21.9. – 29.9.)

Test 1A: Construction of pNorV and norR plasmids

Test 1B: Construction of promoters with esaboxes and esaR plasmids

Test 2: AND gate promoter pNorV+esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

OCTOBER

WEEK 14 (30.9. – 6.10.)

Test 1A: Construction of pNorV and norR plasmids

Test 1B: Construction of promoters with esaboxes and esaR plasmids

Test 2: AND gate promoter pNorV+esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

WEEK 15 (7.10. – 13.10.)

Test 1A: Construction of pNorV and norR plasmids

Test 1B: Construction of promoters with esaboxes and esaR plasmids

Test 2: AND gate promoter pNorV+esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

WEEK 16 (14.10. – 19.10.)

Test 1A: Construction of pNorV and norR plasmids

Test 1B: Construction of promoters with esaboxes and esaR plasmids

Test 2: AND gate promoter pNorV+esabox

Test 3: Switch based on recombinases

Test 4: AND gate promoter pNorV+LldO

Test 5: switch based on Cpf1 system

Directed evolution

General

Thanks to the sponsors that supported our project: