Difference between revisions of "Team:Brasil-USP/Project/RubberDegradation"
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<h1>Rubber Degradation</h1> | <h1>Rubber Degradation</h1> | ||
<p> | <p> | ||
| − | Professor Dieter Jendrossek from Institut für Mikrobiologie, Universität Stuttgart, Germany kindly provided | + | Professor Dieter Jendrossek from the Institut für Mikrobiologie, Universität Stuttgart, Germany kindly provided ORFs for the rubber degradation enzymes Lcp and RoxA cloned into pUC9 propagation plasmid. |
| + | Considering the biobrick standard, we had to clone these genes in pSB1C3 vector. For this purpose, we included restrictions sites that contain the biobrick prefix and suffix using primers as mentioned in “Design”. | ||
| + | But, we found an EcoRI site between bases 207 and 211 of RoxA sequence. Then, we had to remove that restriction site from the original DNA sequence of roxA using site-directed mutagenesis. We can observe a positive result in a restriction gel, with a colony came from a transformation using an aliquot digested with DpnI PCR. | ||
| + | Besides, we thought of cloning the lcp in a expression vector, pETSUMO. This vector is facilitates the purification and solubilization. Therewith will be possible to perform enzymatic assays. This will be necessary to obtain enzyme parameters can be helpfull in comparation in vitro tests with our circuit tests. | ||
</p> | </p> | ||
| Line 44: | Line 47: | ||
<p> | <p> | ||
| − | Mutagenesis PCR of RoxA on the last two lanes successful. We can show that the EcoRI restriction site was removed. | + | Mutagenesis PCR of RoxA on the last two lanes successful. We can show that the EcoRI restriction site was removed. </p> |
<a class="manchor" id="roxaresults"></a> | <a class="manchor" id="roxaresults"></a> | ||
| − | <h2>Vector Changes of | + | <h2>Vector Changes of Lcp and RoxA</h2> |
<p> | <p> | ||
| − | We have added restriction sites in lcp and roxA. Both enzymes were cloned in vector | + | 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. </p> |
<div class="fig" style="width: 600px;"> | <div class="fig" style="width: 600px;"> | ||
<center><img src="https://static.igem.org/mediawiki/2015/d/d4/Team-Brasil-USP-PCR-RoxA-psb1c3.jpeg" width="200"></center> | <center><img src="https://static.igem.org/mediawiki/2015/d/d4/Team-Brasil-USP-PCR-RoxA-psb1c3.jpeg" width="200"></center> | ||
| − | + | <p>Figure 2 - roxA mutated gene in pSB1C3</p> </div> | |
| + | |||
| + | |||
<div class="fig" style="width: 600px;"> | <div class="fig" style="width: 600px;"> | ||
<center><img src="https://static.igem.org/mediawiki/2015/3/36/Team-Brasil-USP-PCR-RoxA-LCP.jpeg" width="300"></center> | <center><img src="https://static.igem.org/mediawiki/2015/3/36/Team-Brasil-USP-PCR-RoxA-LCP.jpeg" width="300"></center> | ||
| − | <p>Figure 3 - lcp in pSB1C3; lcp in pETSUMO for protein expression; Mutagenesis PCR of roxA. </p> | + | <p>Figure 3 - lcp in pSB1C3; lcp in pETSUMO for protein expression; Mutagenesis PCR of roxA. </p></div> |
| − | + | <p> | |
| − | < | + | As we can see in these gels, all vector changes were successful.</p> |
| − | As we can see in these gels, all vector changes were successful. | + | |
| − | + | ||
| − | + | ||
<a class="manchor" id="discussionlcproxa"></a> | <a class="manchor" id="discussionlcproxa"></a> | ||
<h2>Discussion</h2> | <h2>Discussion</h2> | ||
<p> | <p> | ||
| − | + | Lcp and RoxA genes have been adapted to the circuit assembly. This parts can have great value to future iGEM teams, that intend integrate rubber degradation in their projects. We intend to continue the circuit assembly of rubber degradation, analyzing the viability of <i>E. coli</i> for this process. We aim to express RoxA an Lcp solubles to verify its feasibility for an industrial process and make the necessary changes to improve a future scale-up of this process. The next steps is a real test, using rubber result of devulcanization tire come from <i>Acidobacillus ferrooxidans</i> treatment. For this aim, will be necessary first conclude the best exportation system and the best promoter to use. | |
| + | |||
</p> | </p> | ||
Latest revision as of 22:34, 20 October 2015
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 the Institut für Mikrobiologie, Universität Stuttgart, Germany kindly provided ORFs for the rubber degradation enzymes Lcp and RoxA cloned into pUC9 propagation plasmid. Considering the biobrick standard, we had to clone these genes in pSB1C3 vector. For this purpose, we included restrictions sites that contain the biobrick prefix and suffix using primers as mentioned in “Design”. But, we found an EcoRI site between bases 207 and 211 of RoxA sequence. Then, we had to remove that restriction site from the original DNA sequence of roxA using site-directed mutagenesis. We can observe a positive result in a restriction gel, with a colony came from a transformation using an aliquot digested with DpnI PCR. Besides, we thought of cloning the lcp in a expression vector, pETSUMO. This vector is facilitates the purification and solubilization. Therewith will be possible to perform enzymatic assays. This will be necessary to obtain enzyme parameters can be helpfull in comparation in vitro tests with our circuit tests.
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
Lcp and RoxA genes have been adapted to the circuit assembly. This parts can have great value to future iGEM teams, that intend integrate rubber degradation in their projects. We intend to continue the circuit assembly of rubber degradation, analyzing the viability of E. coli for this process. We aim to express RoxA an Lcp solubles to verify its feasibility for an industrial process and make the necessary changes to improve a future scale-up of this process. The next steps is a real test, using rubber result of devulcanization tire come from Acidobacillus ferrooxidans treatment. For this aim, will be necessary first conclude the best exportation system and the best promoter to use.