Team:Brasil-USP/Project/RubberDegradation
Rubber Degradation
Results
Table of contents
Natural and synthetic rubber degradation has been reported for some microorganisms such as Streptomyces sp. strain K30, Gordonia polyisoprenivorans, Nocardia sp. strain 835A and Xanthomonas sp. strain 35Y.1 However, most of these organisms show a slow growth when using rubber as a sole carbon source.2-4
To improve microbial rubber degradation efficiency, our project aims to create a gene circuit which will, among other things, be responsible for coding and expressing two enzymes: RoxA (Rubber oxygenase A) and Lcp (Latex clearing protein). These enzymes are fundamental for rubber degradation and have to be secreted or exposed in the exterior of the cell to be in direct contact with their substrate, the poly(cis-1,4 isoprene) - the main component of rubber. The proposed circuit provides the secretion of the proteins by the fusion with a specific signal sequence, the Twin-arginine translocation (TAT) sequence, or its binding to the bacterial outer membrane by the fusion with a specific protein, OmpA fused with a linker (BBa_K1489002).
Rubber Degradation
Professor Dieter Jendrossek from Institut für Mikrobiologie, Universität Stuttgart, Germany kindly provided the ORFs of the rubber degradation enzymes Lcp and RoxA, in pUC9 propagation plasmid. The first step was remove a EcoRI restriction site from the original DNA sequence of roxA, using mutagenesis PCR. We can observe a positive result in a restriction gel, with a colony came from a transformation using an aliquot digested with DpnI PCR
Mutagenesis PCR of RoxA
Figure 1 - pUC9::roxA has 3703bp; roxA has 2037bp
Mutagenesis PCR of RoxA on the last two lanes successful. We can show that the EcoRI restriction site was removed.
Vector Changes of LCP and RoxA
We have added restriction sites in lcp and roxA. Both enzymes were cloned in vector PSB1C3 to facilitate the insertion in the main circuit. Moreover, the lcp was cloned into the expression vector (pETSUMO) to permit activity assays.
Figure 2 - roxA mutated gene in pSB1C3
Figure 3 - lcp in pSB1C3; lcp in pETSUMO for protein expression; Mutagenesis PCR of roxA.
As we can see in these gels, all vector changes were successful.
Discussion
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