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 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

AUGUST

WEEK 6 (3.8. – 9.8.)

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 7 (10.8. – 16.8.)

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 8 (17.8. – 23.8.)

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 9 (24.8. – 30.8.)

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

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: