Team:Brasil-USP/Project/Results

Results

Table of contents

• Promoter Test
• DH5 alpha
• BL21 (DE3)
• Discussion
• Exportation
• TAT signal
• OmpA
• Discussion
• Rubber Degradation
• Lcp
• RoxA
• Discussion
• Main Circuit

    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).

Promoter Test

The Gram negative bacterium Escherichia coli is the most used chassis in Synthetic Biology. The bacterial system is often chosen due to its low cost, easy maintenance, high productivity and low time consumption. Moreover, there are several large scale system established for E. coli use in industry and, since our project aim for industrial applications, we have chosen the E. coli system allied with the previously named secretion routes that might help increase enzymes' stability. However, the choice of the promoter system is not as simple as the chassis'. That being said, we designed circuits to test the viability of three different inducible promoters: Plac, Para and Prha (figure X).

TAT signal

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Figure 2 - .

OmpA

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Discussion

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Exportation Test

The Gram negative bacterium Escherichia coli is the most used chassis in Synthetic Biology. The bacterial system is often chosen due to its low cost, easy maintenance, high productivity and low time consumption. Moreover, there are several large scale system established for E. coli use in industry and, since our project aim for industrial applications, we have chosen the E. coli system allied with the previously named secretion routes that might help increase enzymes' stability. However, the choice of the promoter system is not as simple as the chassis'. That being said, we designed circuits to test the viability of three different inducible promoters: Plac, Para and Prha (figure X).

DH5 alpha

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Figure 2 - .

BL21 (DE3)

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Discussion

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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

Lcp

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Figure 2 - .

RoxA

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Discussion

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