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Revision as of 17:44, 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.

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: