Difference between revisions of "Team:TU Darmstadt/Engineering"
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Huang et al. already showed that <b>fusion proteins consisting of TasA and another protein</b> (e.g. mCherry or SpyTag proteins) are <b>successfully exported into the biofilm matrix</b>. Even a fusion protein of TasA with the enzyme MHETase (63.1 kDa), that is bigger than the enzymes we want to use, was localized in the biofilm matrix of <i>Bacillus subtilis</i>.<br> | Huang et al. already showed that <b>fusion proteins consisting of TasA and another protein</b> (e.g. mCherry or SpyTag proteins) are <b>successfully exported into the biofilm matrix</b>. Even a fusion protein of TasA with the enzyme MHETase (63.1 kDa), that is bigger than the enzymes we want to use, was localized in the biofilm matrix of <i>Bacillus subtilis</i>.<br> | ||
| − | We <b>conceptualized fusion proteins with our degradation enzymes and the protein TasA</b> based on the research of Huang et <i>al </i> The genes of our laccases CotA and CuO are fused to the | + | We <b>conceptualized fusion proteins with our degradation enzymes and the protein TasA</b> based on the research of Huang et <i>al </i> The genes of our laccases CotA and CuO are fused to the 3' end of the <i>tasA</i> gene with a gene fragment encoding a <b>flexible glycine-serine linker</b> (ARGGGGSGGGGS), that was also used by Huang et <i>al. </i><br> |
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<img src="https://static.igem.org/mediawiki/2020/b/b0/T--TU_Darmstadt--Engineering_Success_TasA.svg" alt="figure"> | <img src="https://static.igem.org/mediawiki/2020/b/b0/T--TU_Darmstadt--Engineering_Success_TasA.svg" alt="figure"> | ||
| − | <figcaption id="Figure#">Figure X: The degradation enzymes are displayed in the biofilm matrix as fusion proteins with the matrixprotein TasA. The gene for the fusion protein is integrated into the genome of B. subtilis. TasA is fused to the | + | <figcaption id="Figure#">Figure X: The degradation enzymes are displayed in the biofilm matrix as fusion proteins with the matrixprotein TasA. The gene for the fusion protein is integrated into the genome of B. subtilis. TasA is fused to the enzyme via a flexible glycine-serine linker. </figcaption> |
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Revision as of 08:57, 22 October 2020
Displaying our degradation Enzymes in the Biofilm Matrix - Fusion Proteins with TasA
Based on our research our goal was to degrade toxic compounds in wastewater by enzymes. These enzymes should be stable and work as efficient as possible. Therefore, we imagined different solutions. Besides secreting our enzymes by microorganisms into the wastewater or add purified enzymes, immobilization would be a possible solution. Immobilization can be realized on various materials but also by using biological systems. Huang et al. reached immobilization of proteins and enzymes in the biofilm matrix of Bacillus subtilis (5). They successfully built fusion proteins of the matrix protein TasA with other proteins or enzymes (5).
Considering the manifold advantages of immobilization (Verlinkung text pharmis) and the research of Huang et al. we decided to display our degradation enzymes in the biofilm matrix of B. subtilis as fusion proteins with TasA (Verlinkung Text Filmis). The matrix protein TasA fits best as a fusion protein because it is a main compound of the biofilm matrix leading to a high number of enzymes displayed. By adjusting the fusion proteins, we could display different enzymes, making our project more modular (Link zu modularity text?). Since we use a living system, we avoid elaborate purification of enzymes and can easily adapt our biofilm.
We conceptualized fusion proteins with our degradation enzymes and the protein TasA based on the research of Huang et al The genes of our laccases CotA and CuO are fused to the 3' end of the tasA gene with a gene fragment encoding a flexible glycine-serine linker (ARGGGGSGGGGS), that was also used by Huang et al.
Cells of the B. subtilis strain GP1622 will be transformed with this plasmid using a protocol obtained from Prof Stülke (Georg-August-Universität Göttingen) (7). The strain also obtained from Prof. Stülke lacks the genes sinR and tasA (7). With the sinR knockout we improve the biofilm formation (Verlinkung Text Filmis und quelle) and the tasA knockout makes sure that only our fusion proteins are integrated into the biofilm matrix (8) (Verlinkung Text filmis).
As a proof of concept, we built a fusion protein of TasA and superfolder green fluorescent protein (sfGFP) . The sequence of the sfGFP gene is codon optimized for B. subtilis and fused to tasA as described above. The display of the TasA-sfGFP fusion protein in the biofilm matrix can be verified using fluorescence microscopy. Therefore, we would use the measurement kit of iGEM to standardize our results.
We hypothesize that TasA fusion proteins with our targeted degradation enzymes will succeed, if TasA-sfGFP is successfully expressed in the biofilm matrix.
Due to the Covid-19 pandemic we were not able to conduct our experiments in the laboratory. In the following we discuss possible outcomes and possible adaptions of our experimental setup.
If our biofilm is built correctly but we are unable to measure any fluorescence we can conclude that there has to be a problem with the sfGFP. It can mean that it was not even expressed or lost its function due to the fusion with TasA. To react on the former, we would check the sequences of our ordered sfGFP-TasA gblock as well as doing a plasmid preparation with following sequencing of our PCR amplified fusion protein parts. Approaching to a possible loss of function due to the fusion with TasA we would try to use a different linker. A potential reason for the loss of function can be too less space between the proteins, leading to misfolding or aggregation. This can be solved with a longer and more rigid linker (9). Another reason could be that sfGFP cannot be folded fast enough before secretion into the matrix with TasA. This can also be adapted by changes in the linker sequence (10). We would try to use amino acids that slow down the translation of the fusion protein, for example proline. By testing different protein architectures, we can learn more about the TasA-fusion platform in general, helping other researchers who also want to use this technology.
If the biofilm we grow seems to be less stable and we are able to detect fluorescence in our cells the export of TasA-sfGFP into the biofilm matrix is detracted by the fusion of TasA to sfGFP. In this case we would redesign our fusion protein especially regarding to the used linker as well as checking the secretion sequence for the transport of TasA.
A less stable biofilm combined with lack of any fluorescence could be explained with an unsuccessful transformation of our B. subtilis cells, a mistake in the sequence of our fusion protein or a mistake in our plasmid. Promoter, ribosome binding site or selection marker could be defective.
Further steps would be the repetition of the described experiment with TasA-CueO and TasA-CotA fusion proteins. The successful display of these enzymes in the biofilm matrix could be verified using the ABTS assay (Verlinkung pharmis). Furthermore, for the implementation of our biofilm the gene for our fusion proteins should be integrated into the genome of the B. subtilis strain via homologous recombination using integrative vectors (11).
Experts
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Former iGEMers
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Others
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