Team:GO Paris-Saclay/Parts/CPG2

Context

An enzyme that cleaves folates, yielding a pterydoyl ring and glutamate(s).

CPG2 activity has been previously demonstrated by HPLC analysis after 5 h and 20 h incubation of MTX with bacteria harbouring pSB1C3-cpg2 (BBa_K2688003). As previously mentioned by the team that cloned and characterized this biobrick, the “earliest sampling (at 5 h) has been shown by HPLC to be “too late” (almost all the MTX is already gone from the medium at this time point).” To gain new insights on this aspect, we decided to perform a kinetic analysis of MTX biotransformation by a bacteria expression cpg2.

Materials and methods

E. coli BW25113 strains harbouring pSB1C3-cpg2 (BBa_K2688003) or pSB1C3-tet (BBa_R0040) were grown in LB containing 0.5 mM IPTG and 0.2 % (m/v) arabinose (same conditions than previously used by the first team that characterized BBa_K2688003). The biotransformation experiments were conducted in 15 mL Falcon tubes, with 1 mL of the same medium supplemented with 100 µM MTX, inoculated with the strains at an initial OD 600nm of 0.2. The tubes were incubated under agitation at 37°C. Two hundred µl were harvested after 1 h and 3 h incubation with bacteria. Samples were filtered (0.2 µm) and submitted to HPLC analysis performed using a reverse phase C18 column as previously described (see the detailed protocol of HPLC analysis method above). Pure 100 µM MTX and DAMPA were used as controls for HPLC analysis.

Results

Figure legend: Kinetic analysis of MTX biotransformation. Bacteria were incubated in LB medium containing 100 µM MTX (see the M&M for details). LB media without bacteria containing MTX or DAMPA was used as a control. DAMPA control was performed at the beginning of the experiment (time point 0). Results were normalized on the percentage of MTX control condition.

Result summary and perspective

We observed that bacteria expressing cpg2 are able to remove efficiently 100 µM MTX from LB media in 3 h. We managed to catch intermediate conditions in which MTX is still detected. After 1 h, 70 % of the MTX were biotransformed into DAMPA. Bacteria expression cpg2 are able to remove rather rapidly (in a few hours) 100 µM MTX for the media.

We also tested the biotransformation of MTX by CPG2 in a DNA-less chassis strain (see the DNA-free POETential project - https://2019.igem.org/Team:GO_Paris-Saclay).

Contacts

Email: igem.parissaclay@gmail.com

2019 GO Paris Saclay Team

We are proud to present our project to all our IGEM friends :)

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