Difference between revisions of "Team:ETH Zurich/Notebook"

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

Revision as of 17:46, 18 October 2016

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.)

WEEK 4 (20.7. – 26.7.)

WEEK 5 (27.7. – 2.8.)

AUGUST

WEEK 6 (3.8. – 9.8.)

WEEK 7 (10.8. – 16.8.)

WEEK 8 (17.8. – 23.8.)

WEEK 9 (24.8. – 30.8.)

SEPTEMBER

WEEK 10 (31.8. – 6.9.)

WEEK 11 (7.9. – 13.9.)

WEEK 12 (14.9. – 20.9.)

WEEK 13 (21.9. – 29.9.)

OCTOBER

WEEK 14 (30.9. – 6.10.)

WEEK 15 (7.10. – 13.10.)

WEEK 16 (14.10. – 19.10.)

Thanks to the sponsors that supported our project: