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).

DH5α

We first started with DH5α. Spoiler alert: we didn't see very promising results. We evaluated the fluorescence per cell by measuring Optical Density (OD) of our colonies and their fluorescences. We show in figures 1 and 2 our results.

Figure 1 - Optical Density (OD) measurements over time, showing that all colonies were in their log phase.

Figure 2 - Fluorescence per cell for each promoter, normalized by the fluorescence with no induction. In the x-axis, the higher the concentration label, the higher the concentration, with zero means no induction at all. Only Rhamnose showed some actual induction.

Except for Rhamose, fluorescence was very weak and basically at the level of our negative control (E. Coli without plasmid), confirmed by a Mann-Whitney U test. Rhamnose showed some level of activity which would be considered weak, compared to, for instance, our Interlab Results. Finally, no considerable changes were detected in the RFP measures.

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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References

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2. Rose, K., Tenberge, K. B. & Steinbuchel, A. Identification and characterization of genes from Streptomyces sp strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation. Biomacromolecules 6, 180-188, doi:10.1021/bm0496110 (2005).
3. Tsuchii, A. & Takeda, K. Rubber-degrading enzyme from a bacterial culture. Appl. Environ. Microbiol. 56, 269-274 (1990).
4. Bode, H. B., Kerkhoff, K. & Jendrossek, D. Bacterial degradation of natural and synthetic rubber. Biomacromolecules 2, 295-303, doi:10.1021/bm005638h (2001).
5. Mergulhão, F. J. M.; Summers, D. K.; Monteiro, G. A. Recombinant protein secretion in Escherichia coli. Biotechnology Advances. 23, 177–202 (2005).
6. Delepelaire, P. Type I secretion in gram-negative bacteria. Biochimica et Biophysica Acta. 1694, 149–161 (2004).