Team:UANL Mty-Mexico/Wet lab/Integration

Due to complexity of the circuit for giving capacity to this cells to respond to the light code, photoreceptor induction system was planed to be integrated into the E. coli genome, this way, there would be constructed three new different E. coli modified strains which have the capacity of light responding, one to red light (E. coli MX^Red), other to green light (E. coli MX^Green), both used in the HuBac community and a third extra cell which would be able to respond to both lights (E. coli MX^RedGreen) as explained in the section Photochassis.

Integration protocol was performed according to the technique of site-specific chromosomal integration of large synthetic constructs developed by Thomas E. Kuhlman and Edward C. Cox[1]. Integration protocol was modified for this project as detailed in the section Notebook: Protocols and, briefly, it consists on next steps:

1. Transforming cells with helper plasmid pTKRED
2. Transforming cells with PCR product of Landing Pad (from pTKS/CS plasmid) which is meant to be integrated into the E. coli genome through λ-Red enzyme induced with IPTG.
3. Transforming cells with pTKIP plasmid which contains the desired fragment to be inserted.
4. Recombination of Landing Pad and the fragment to be inserted, this would be through λ-Red and I-SceI enzymes, respectively induced with IPTG and arabinose.
5. Plasmid curation.

Characterization of pTKRED transformed into E. coli JT2 strain

PCR of Landing Pad from pTKS/CS plasmid

Characterization of Landing Pad PCR product.

Digestion of Landing Pad with DpnI enzyme previous to electroporation

Cells transformed with Landing Pad were selected by antibiotic resistance and then PCR from genomic DNA extraction was performed, sample M2 was the only positive result, it was named E. coli MX1

After demonstrating that Landing Pad could be amplified from genomic DNA extraction, an additional experiment was performed in order to verify absence of chloramphenicol resistance and presence of tetracycline resistance, this to eliminate possibility of plasmid transformation (pTKS/CS which represents template for Landing Pad amplification contains chloramphenicol and tetracycline resistance inside Landing Pad, though, only real non-integrated cells would survive on tetracycline or chloramphenicol supplemented media). Next figure shows a replicate plate test for E. coli MX1 cells on both antibiotics of interest.

Replicate plate test for E. coli MX1 cells on tetracycline and chloramphenicol. Positive control for chloramphenicol resistance are highlighted on red boxes.

After transforming E. coli MX1 cells with pTKIP-hph plasmid, positive colonies were selected for recombination step on integration protocol. Cells were plate on LB agar supplemented with hygromycin and then used for replicate plate test against ampicillin, this for looking positive recombinants.

Transformed MX1 cells with pTKIP-hph plasmid were selected for replicate plate test. Left) LB agar supplemented with ampicillin, Right) LB agar agar supplemented with hygromycin. No positive recombinants were found.

After having not positive recombinant colonies in the recombination step, different experiments were made in order to test different factors such as media, arabinose concentration, incubation time and conditions. No significant results were obtained. (Data not shown).

1. Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res 38:e92.