Composite

Part:BBa_K4989011

Designed by: Athanasia Arampatzi   Group: iGEM23_Thrace   (2023-10-12)
Revision as of 12:08, 12 October 2023 by Atharab (Talk | contribs) (Configuration of the new part)

Complete butyrate producing gene cluster

Short Description

In this registry page, we will present our New Improved Part that our team created to be nominated for the gold medal in the iGEM 2023 competition! We designed a whole composite part, with its coding and regulatory sequences, that can potentially produce butyrate, a Short Chain Fatty Acid (SCFA) that could potentially treat depression.


The fundaments of the improvement

To result in the design of our new improved part, we needed to find a corresponding part that requires such changes that would not change the function of the part and the general aim of its initial formation but enhance the overall performance.
Team NRP-UEA-Norwich 2015 designed the BBa_K1618021 composite part that could potentially produce butyrate. We reviewed the part and we realized that it is our ideal candidate to enhance its features and produce a stronger gene cluster of the butyrate production pathway.
Simultaneously, we conducted extensive bibliographic research and we created a draft of the ideal gene cluster and its regulatory elements. In this way, we could compare the ideal draft of the part that we envisioned with the part that was pruned to be improved.



Application in the field of biology

Short-chain fatty acids are vital metabolic byproducts generated through microbial fermentation of dietary fibers in the gut. Gut bacteria, particularly those belonging to the Firmicutes and Bacteroidetes phyla, break down these substrates through various metabolic pathways. There are several fermentation pathways leading to the production of SCFAs, including the glycolytic pathway, the pentose phosphate pathway, and the reductive citric acid cycle. In these pathways, bacteria metabolize sugars and other organic compounds to produce SCFAs as metabolic byproducts.

Acetate Production: Acetate is the most abundant SCFA in the gut. It is primarily generated through the acetyl-CoA pathway, where acetyl-CoA is converted into acetate via various enzymatic reactions.
Propionate Production: Propionate is synthesized mainly via the succinate pathway. Bacteria convert succinate, a metabolic intermediate, into propionate. This pathway is essential for maintaining energy balance and regulating blood glucose levels.
Butyrate Production: Butyrate is generated through several pathways, with the two main pathways being the butyryl-CoA:acetate CoA-transferase pathway and the butyrate kinase pathway. These pathways involve the conversion of acetyl-CoA and butyryl-CoA into butyrate.

From bibliographical research and various experimental data, we concluded that acetate and butyrate are the most profound and effective molecules for the treatment of depression, and generally the overall gut health. Therefore, the design of a probiotic that would include a vector with the gene cluster of the butyrate-producing metabolic pathway, could lead to the production of a psychobiotic that could treat depression. Our selected strain Lactobacillus rhamnosus GG produces only acetate.Lactobacillus rhamnosus GG is a very common probiotic that is Generally Recognised As Safe (GRAS) and our dry lab bioinformatic analysis found that the concentration of the Lactobacillus rhamnosus bacterium is low on depressive people. Thus it made us focus on this probiotic for its utilization as a host of the butyrate-producing pathway.


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Pathways for the synthesis of SCFAs. Lange, O., Proczko-Stepaniak, M. & Mika, A. Short-Chain Fatty Acids—A Product of the Microbiome and Its Participation in Two-Way Communication on the Microbiome-Host Mammal Line. Curr Obes Rep 12, 108–126 (2023). https://doi.org/10.1007/s13679-023-00503-6

Configuration of the new part

In this section, we will shortly analyze the re-design process of our part. The extensions of this process and the more detailed handling are analyzed in our team's New Improved Part Wiki section (see at the end of the page).
The re-design project was performed on 3 separate levels:

A. On a sequence level
B. On a regulatory level
C. On a structural level

On a sequence level, we needed to change the whole DNA makeup of the genes and codon-optimize them to be properly expressed in the corresponding hosts.

On a regulatory level, we added some very crucial regulatory elements to ensure the expression of the enzymes. We included the P32 promoter with its RBS for the expression to be compatible with our target host Lactobacillus rhamnosus GG. Also, we added at the of the part a terminator sequence specifically designed for Lactobacillus spp..In this way, we ensured our transcriptional termination. Lastly, we included in-between the coding sequences Ribosome Binding Sites, in order for one transcriptional mRNA to be produced by all the target enzymes of the pathway.

On a structural level, we maintained the order of the genes and we added, upon prompt from our dry lab bioinformatic analysis, the butyryl-CoA:acetyl-CoA transferase that catalyzes the last step of the butyrate production.

Our new improved part has the characteristic of a whole constructed transcriptional cassette. However, we clearly have marginalized all the basic parts (components) in order for the P32 promoter and the terminator to be removed, always based on the regulatory feature that the cloning vector has.

The obtainment of the sequence and its difficulties

Biosafety

Characterization

References

External links

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