Part:BBa_K4989011:Design
Complete butyrate producing gene cluster
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1143
Illegal BglII site found at 6852
Illegal BamHI site found at 1008 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 291
Illegal NgoMIV site found at 2703
Illegal NgoMIV site found at 6067
Illegal AgeI site found at 1032
Illegal AgeI site found at 1066
Illegal AgeI site found at 5423
Illegal AgeI site found at 7026 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
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. Also, we performed an in-silico optimization of the sequence to not include any restriction sites of the endonucleases that we intended to use.
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, 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 have marginalized all the basic parts (components) for the P32 promoter and the terminator to be removed, always based on the regulatory feature that the cloning vector has.
Source
Our initial goal was to synthetically produce the whole sequence. But might be difficult due to the fact that the sequence is large in size and unfortunately, it includes a high rate of complexity. Also, synthesizing the terminator might not be able to happen due to the hairpin that it forms. Twist Bioscience, one of this year's sponsors synthesized our part in gBlocks that we later assembled with the GoldenGate method.
The sequences where obtained from basic parts already recorded in the registry (P32,terminator,RBS), from Coprococcus sp. L2-50 DSM (all the genes up to Eftα) and from Roseburia intestinalis L1-82 (butyryl-CoA:acetyl-CoA transferase).
References
[1]Berezina, Oksana V., et al. “Reconstructing the Clostridial N-Butanol Metabolic Pathway in Lactobacillus Brevis.” Applied Microbiology and Biotechnology, vol. 87, no. 2, Mar. 2010, pp. 635–46, https://doi.org/10.1007/s00253-010-2480-z. Accessed 26 Oct. 2021.
[2]Wang, Liang, et al. “Metabolic Engineering of Escherichia Coli for the Production of Butyric Acid at High Titer and Productivity.” Biotechnology for Biofuels, vol. 12, Mar. 2019, https://doi.org/10.1186/s13068-019-1408-9. Accessed 8 Apr. 2021.
[3]Petra Louis, Sheila I. McCrae, Cédric Charrier, Harry J. Flint, Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer, FEMS Microbiology Letters, Volume 269, Issue 2, April 2007, Pages 240–247,https://doi.org/10.1111/j.1574-6968.2006.00629.x
[4]“Butyrate-Producing Bacterium L2-50 Putative Fe-S Oxidoreductase Gene, Partial Cds; Thiolase, Crotonase, Beta Hydroxybutyryl-CoA Dehydrogenase, Butyryl-CoA Dehydrogenase, Electron Transfer Flavoprotein Beta-Subunit, and Electron Transfer Flavoprotein Alpha-Subunit Genes, Complete Cds; and Putative Multidrug Efflux Pump Gene, Partial Cds.” NCBI Nucleotide, July 2016, https://www.ncbi.nlm.nih.gov/nuccore/DQ987697.1/
[5]Roseburia Intestinalis L1-82 Chromosome 1, Complete Sequence.” NCBI Nucleotide, Mar. 2023, www.ncbi.nlm.nih.gov/nuccore/NZ_LR027880.1?report=genbank&from=430426&to=431769&strand=true. Accessed 12 Oct. 2023.