Team:UANL Mty-Mexico/Wet lab/Integration

To overcome the difficulties of handling large and numerous extra-chromosomal molecules, we planned to integrate the genetic light induction system into the E. coli chromosome. Three new strains are to be created, as described in the Photochassis section. We dubbed these modified strains according to the light they respond as follows:

• MXred, which responds to red light.
• MXgreen, which responds to green light.
• MXredgreen, which responds to both, red and green lights. This one does not make part of the community, however we believe it could be useful in the light induction field.

Integration protocol was performed through the method of site-specific chromosomal integration of large synthetic constructs, developed by Thomas E. Kuhlman and Edward C. Cox[1]. The protocol was modified for this project as detailed in the section Notebook: Protocols. Briefly, the integration method consists on the following 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 IPTG induced recombineering.
3. Transforming cells with pTKIP plasmid, which contains the fragment to be inserted.
4. Recombineering of the Landing Pad and the fragment to be inserted through λ-Red and I-SceI, induced with IPTG and arabinose respectively.
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.