Improvement on Existing Parts
Unfortunately due to the lack of lab access available to the St Andrews iGEM team this summer, no physical lab work could be carried out to physically improve previous parts on the registry. Despite this, many in silico means were taken to meet this criteria with these improvements falling under three major categories:
- Codon Optimisation of Pre-existing Registry Parts
- Description Update for Pre-existing Registry Parts
- Computational simulation data for Pre-existing Registry Parts
Codon Optimisation of Pre-existing Registry Parts
Initially when the idea of a probiotic sunscreen was proposed, the St Andrews iGEM team looked to source a molecule with UV-absorbing properties and which was produced naturally by an organism. Stumbling upon mycosporine-like amino acids (MAAs), we realised that these natural UV-absorbing compounds were produced by an array of different aquatic species including cyanobacteria and red algae.
We also considered the chassis to work with and selected E.coli Nissle 1917 given its ease of genetic transformation, rate of proliferation and associated lack of pathogenecity. In order to improve transcriptional and translation efficiency however, we needed an organism that was as closely related the chassis organism as possible.
For this, we selected the shinorine gene cluster from Anabaena variabilis, a species of filamentous cyanobacteria from the Eubacteria domain. Although selecting the gene cluster from a cyanobacteria capable of producing these UV-absorbing compounds over an algae would undoubtfully improve enzyme expression efficiency, previous research by Balskus and Walsh (2010) showed the total mass of shinorine and other MAAs to be much less when compared to other bacterial hosts (Tsuge et al., 2018) thus making the E.coli chassis a problem.
Methods of improving transcriptional and translation efficiency were therefore investigated. Alongside gene knockout to improve metabolic flux of the substrate sedoheptulose 7-phopshate to the shinorine pathway, codon optimisation of the genes for the shinorine-generating enzymes was discussed. Agreeing on this technique to improve shinorine production, codon optimisation was achieved by using the IDT codon optimisation tool (https://eu.idtdna.com/CodonOpt). All parts non-native to E.coli besides the highly specific R.CviJI endonuclease component were codon optimised. Below is a table summarising the pre-existing parts taken and the new E.coli codon optimised parts. For some optimised parts, further in silico site-directed mutagenesis was carried out as to remove illegal BioBrick standard restriction sites introduced by the IDT codon optimisation tool.
|Part Name||Pre-existing BioBrick||Codon Optimised Replacement|
|3-Dehydroquinate Synthase (DHQS)||BBa_K814000||BBa_K3634000|
|Nonribosomal Peptide Synthetase (NRPS)||BBa_K814003||BBa_K3634003|
|Heme Oxygenase (ho1)||BBa_K2328062||BBa_K3634007|
|Phycocyanobilin:Ferredoxin Oxidoreductase (pcyA)||BBa_I15009||BBa_K3634008|
It had previously been shown by Minnesota iGEM (2012) that the final enzyme in the reaction, NRPS, was the rate-limiting step of shinorine formation. It was proposed that, by carrying out the required codon optimisation step, the efficiency of the enzyme would be increased due to greater intracellular concentrations of NRPS available. This assumption will be tested in phase II of the project (2021).
Description Update for Pre-existing Registry Parts
As the idea surrounding our final synthetic gene circuit evolved throughout the months of June and July, a lot of research was carried out into both the shinogen and thanogen aspects of the circuit. On top of the information found from academic papers and references, the St Andrews iGEM team 2020 sought to find any pre-existing parts which we could utilise and transform into our organism.
The registry of standard biological parts is a fantastic resource for previous research and experimental data on thousands of biological systems. Somewhat disappointingly, only those parts that have been used tens to hundreds of times have been satisfactorily characterised on their respective pages by the teams that have contributed to them.
When researching our circuit, aspects such as the light sensor, toxin/antitoxin modules (TA) and the shinorine pathway had all been uploaded before but only a limited extent of information was provided. Alongside creating new codon optimised parts, we began uploading to previous constructs our new part contribution alongside a variety and depth of literature that the team had obtained through review articles and published research. Updated information was provided for the following parts (N.B. the UirS/UirR system (BBa_K1725400 – BBa_K1725420) discontinued in our current line of research was studied extensively and thus a contribution felt necessary):
An in-silico gel was synthesised using SnapGene to present the standardisation of all parts in the plasmid. This showed that no additional cut fragments were present.
Figure 1. Molecular weights of pSB3E1-AN (lanes 1,2,3,4), undigested (lane1), digested with R.CviJI (lane2), RFC illegal endonucleases (lane3) and RFC illegal endonucleases (lane4). Molecular weights of pSB3B1-DOPH (lanes 6,7,8,9), undigested (lane6), digested with R.CviJI (lane7), RFC illegal endonucleases (lane8) and RFC illegal endonucleases (lane9). Lane 5 was left blank.