Team:UANL Mty-Mexico/Wet lab/Integration
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- | <p> | + | <p>To overcome the difficulties of handling large and numerous extra-chromosomal molecules, we planned to integrate the genetic light induction system into the <i>E. coli</i> chromosome. Three new strains are to be created, as described in the <a href="https://2011.igem.org/Team:UANL_Mty-Mexico/Contributions/Photochassis" title="Photochassis">Photochassis</a> section. We dubbed these modified strains according to the light they respond as follows: |
- | <p>Integration protocol was performed | + | <ul> |
+ | <li>MXred, which responds to red light.</li> | ||
+ | <li>MXgreen, which responds to green light.</li> | ||
+ | <li>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.</li> | ||
+ | </ul> | ||
+ | <p>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<a href="#References" class="references-link">[1]</a>. Integration protocol was modified for this project as detailed in the section Notebook: Protocols and, briefly, it consists on next steps:<ol> | ||
<li>Transforming cells with helper plasmid pTKRED</li> | <li>Transforming cells with helper plasmid pTKRED</li> | ||
<li>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.</li> | <li>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.</li> |
Revision as of 23:17, 28 September 2011
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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]. Integration protocol was modified for this project as detailed in the section Notebook: Protocols and, briefly, it consists on next steps:
- Transforming cells with helper plasmid pTKRED
- 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.
- Transforming cells with pTKIP plasmid which contains the desired fragment to be inserted.
- 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.
- Plasmid curation.
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.
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.
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).
Integration procedure | ||
---|---|---|
Steps | Status | |
Transforming with helper plasmid pTKRED | Done | |
Integration of Landing Pad | Done | |
Transforming with pTKIP plasmid. | Done | |
Recombination of pTKIP plasmid with Landind Pad. | In progress |
- Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res 38:e92.