Team:Westminster UK/Design
Project
Construct design
As a rationale for our design, we used the Mtr pathway for reference. The diagram below demonstrates the overview of the basic parts we sequenced, with Mtr components being solely created for a sequence upgrade for already existing characterised Mtr components available in the registry (http://parts.igem.org/Special:Search?search=Mtr) and coloured parts being separate components we mainly focused on characterising.
Figure 1. Overview of synthesised parts including Mtr components A-B-C and OmcA that form natural complexes, RhlA, the biosurfactant production protein found in Pseudomonas aeruginosa and transcription factors, EtrA, CRP, RpoE that are involved in anaerobic regulatory response.
Constructs were initially designed through Benchling and the final products were adjusted through geneious, our sponsor software. Constructs snapshots seen below were obtained from geneious (find the link for software at the footer of the page).
First stage: RFC adjustment
We chose to modify the prefix and suffix ends of our sequences with RFC25 hangovers, which would allow us to have more versatile sequences capable of being bound to reporter sequences without dysfunctional scar sites (which appear
in RFC10).
RFCs function as part compatibility insurance. They are put onto the ends of a part sequence and contain the necessary cutting sites for restriction enzymes.
Our motivation to switch from using RFC10, the widely adopted assembly standard in synthetic biology, to RFC25 was driven by the support for genetic sequence combinations of parts with linker domains. RFC25 was designed and introduced in 2007 as an extension of the BioBrick RFC[10] that allows for in-frame protein assembly, while also avoiding some concerns with the Silver RFC[25].
Scar resulting from a ligation between two RFC25 parts goes [part A] ACCGGC [part B], which contains two codons for threonine and glycine, making the sequence completely readable.
Important note: in August we checked the site again and found out that iGEM has depracated RFC25, along with 12, 21 and 23. This means that they still acknowledge it, however it is not recommended to be used. Since we already had our sequences and our time was limited enough already with delivery delays due to unforeseen circumstances, we decided to proceed with the sequences we had since they are still characterisable and work almost the same as RFC10.
In future, we recommend using whether RFC10 (in cases where no in-frame assembly is necessary) and RFC1000 (TYPEIIS) in cases where multiple sequences should be aligned together.
Figure 2: Our main software for initial construct design was Benchling - a highly versatile online software also recommended by iGEM.
Part design rationale
We synthesised four new sequences for MtrA, MtrB, MtrC and OmcA components with new RFC adjustments. We recommend using these if your characterisation motive strictly includes the use of RFC25. Otherwise refer to Mtr components designed by the westminster 2015 iGEM team (https://2015.igem.org/Team:Westminster/Parts).
Our characterisations were mainly focused on discovery within the areas of Mtr component transcription regulation and activity.
Since this area is under heavy research momentarily, we looked through the latest research to find best clues into the most promising modifications we could be focusing on.
A research by Barchinger et al (2016) identified
gene transcription changes in anaerobic conditions compared to aerobic conditions that were responsible for the upregulation of mtr pathway components, that would ultimately result in the promotion of cytochrome production and
localisation to the inner and outer membrane of the cell.
Figure 3: The proposed rationale behind the overexpression of transcription factor components. Black arrows are indicating the concentration change, while the colored arrows are referring to the regulation of the promoter, with green (up arrows) indicating increased expression and red (down arrows) indicating decreased expression.
The other gene we identified is rhamnolipid A (RhlA) which codes for the protein 3-(3-hydroxydecanoyloxy) decanoate synthase identified in Pseudomonas aeruginosa strain 1C. It was found to be required for rhamnolipid surfactant production, by supplying acyl moieties for rhamnolipid biosynthesis by competing with the enzymes of the type II acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. We are observing it since its increased presence was found to promote electricity generation from MFCs (Zheng et al, 2015) and are hoping to identify similar effects in Shewanella. If Shewanella appears to not contain the genetic circuit for the relevant pathways, other teams working with Pseudomonas a. can use it for a similar purpose, since its BioBrick was not available in the registry prior to our contribution.
References
Barchinger, S. E., Pirbadian, S., Sambles, C., Baker, C. S., Leung, K. M., Burroughs, N. J., Golbeck, J. H. (2016). Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation. Applied and environmental microbiology, 82 (17), 5428–5443.
Zheng, T., Xu, Y. S., Yong, X. Y., Li, B., Yin, D., Cheng, Q. W., Yuan, H. R., Yong, Y. C. (2015). Endogenously enhanced biosurfactant production promotes electricity generation from microbial fuel cells. Bioresour Technol, 197, 416-21.
