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.

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.

SAFETY FEATURES OF OUR FINAL PROJECT DESIGN

The final goal of our project is to design a bacteria-based detection system to simultaneously detect compounds associated with inflammation and microbiota in the gut of an inflammatory bowel disease (IBD) patient. This requires ingestion of our device by the patient. Our bacteria will travel through the human digestive system in a capsule. The encapsulated bacteria will be collected from the feces and will be analyzed in the lab. The administration and recovery of our bacterial device would be done by trained medical personnel. There are several safety risks which we need to consider in the design of our final device.

RISK OF INFECTING HUMAN GUT

The strains of E. coli we use have a low, but not non-existent, virulence. There is a non-zero probability of our strains mutating to a pathogenic serotype. The device in our final design would consist of bacteria encapsulated in a material (chitosan or alginate) which does not dissolve in the stomach or in the gut, but would allow exchange of small molecule signals. This would isolate the bacteria in the device from the gut microbiota. That would lower the risk of infecting human gut and prevent our bacteria from disrupting the native microbiota population. Because Escherichia coli have pathogenic serotypes, it is not an ideal organism for an in situ study of human gut. Escherichia coli might also cause additional disruption of microbiome balance in the IBD patients. In our final design we would replace E. coli with Lactobacillus sp. We decided to take this safety precaution after discussing with prof. Rogler

RISK OF HORIZONTAL GENE TRANSFER

Horizontal gene transfer could potentially cause antibiotic resistance of gut microbiota. We would use a capsule to prevent interaction between our designed bacteria and the native microbiota. However, this would not eliminate the risk of horizontal gene transfer, which is why we would have to clone our system into a chromosome to eliminate the need for antibiotic resistance and to maximally reduce the risk of gene transfer. Another option would be to use metabolic complementation instead of antibiotic selection for plasmid maintenance.

RISK OF RELEASE IN THE ENVIRONMENT

The capsule would lower the risk of release in the environment by isolating the bacteria from our device from the environment. Since the capsule can be broken or damaged, we would additionally have to develop a knock-out strain which would rely on a metabolite abundantly present in the gut but not in the environment.

Thanks to the sponsors that supported our project: