Difference between revisions of "Team:Brasil-USP/Project/Results"
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| − | + | The ompA mechanism is inserted in the type I secretion system which, in Gram negative bacteria, promotes the secretion of various proteins of different sizes and functions in one single step, without the necessity of a periplasmic intermediate. The secretion is driven by three proteins of the cell envelope6. The first one is located in the outer membrane, and is called OMP (Outer Membrane Protein); the other two are cytoplasmic membrane proteins, called ABC (ATP-Binding Cassette) and MFP (Membrane Fusion Protein). The protein being transported has a specific C-terminal signal sequence that usually has few to 50 glycine rich-repeats, that might be necessary for the protein function and, therefore, is not cleaved during the secretion6. This signal sequence recognizes the ABC protein and triggers the assembly of the whole complex for the translocation process. | |
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Revision as of 05:02, 18 September 2015
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).
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).
DH5 alpha
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Figure 2 - .
BL21 (DE3)
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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).
TAT signal
The Twin-arginine translocation (TAT) system is classified as part of the type II bacterial secretion mechanism, which is composed of two steps. The first one aims to get the protein through the bacterial cytoplasmic membrane and, for that, it can use three different pathways (the SecB-dependent pathway, the signal recognition particle (SRP), and the twin-arginine translocation (TAT)). The second step is the protein translocation through the outer membrane, involving specific protein machinery known as the secreton.5
The TAT pathway, unlike the SecB or the SRP pathways, is capable of transporting folded proteins across the inner membrane independently of ATP using the transmembrane PMF (proton-motive force).5 This translocation system is composed mainly of five proteins (TatA, TatB, TatC, TatD and TatE) but their specific function have not been firmly established yet. However, it is known that TatA is the most expressed gene of this pathway and it has 60% of homology with TatE, which can partially substitute TatA; TaTB can form a complex with TatC to prevent its degradation; there is evidence that TatC is the signal peptide binding protein and, finally, TatD produces no effect on protein translocation but has DNase activity.5 The secretion is composed by 12-16 different proteins, but none of these proteins have their specific roles elucidated.5
Figure 2 - .
OmpA
The ompA mechanism is inserted in the type I secretion system which, in Gram negative bacteria, promotes the secretion of various proteins of different sizes and functions in one single step, without the necessity of a periplasmic intermediate. The secretion is driven by three proteins of the cell envelope6. The first one is located in the outer membrane, and is called OMP (Outer Membrane Protein); the other two are cytoplasmic membrane proteins, called ABC (ATP-Binding Cassette) and MFP (Membrane Fusion Protein). The protein being transported has a specific C-terminal signal sequence that usually has few to 50 glycine rich-repeats, that might be necessary for the protein function and, therefore, is not cleaved during the secretion6. This signal sequence recognizes the ABC protein and triggers the assembly of the whole complex for the translocation process.
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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Main Circuit
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