Team:St Andrews/Contribution

Contribution

Shinogen – the Shinorine-producing system: Last year, the St Andrews’s 2020 (Phase I) iGEM team left us a series of Biobricks, these being Part:BBa_K3634000 – Part:BBa_K3634021. Some of these parts (Part:BBa_K3634000 – Part:BBa_K3634005) were involved in the Shinorine-producing system, Shinogen: the parts for ATPG, NRPS, DHQS, and O-MT respectively (the enzymes in the shinorine-producing pathway) were parts Part:BBa_K3634000 - Part:BBa_K3634003. Part:BBa_K3634004 and Part:BBA_K3634005 were composite parts for DHQS and O-MT, and ATPG and NRPS respectively. These composite parts became redundant during Phase II of our project since it turned out that they were too complex to be manufactured by IDT, and moreover the plasmids we intended to use (pETDuet-1 and pRSFDuet-1) had their own cloning sites with their own promoters, ribosome binding sites (RBS), and terminators – hence we did not need the composite parts containing extra promoters, RBSs and terminators for each of the genes – we decided to use the codon-optimised genes created by last year’s team (Part:BBa_K3634000 – Part:BBa_K3634003) instead, simply inserting these into the cloning sites of our plasmids.

However, upon trying to order the parts for ATPG, NRPS, DHQS, and O-MT (Part:BBa_K3634000 - Part:BBa_K3634003) from IDT, it was found that Part:BBa_K3634003 (which coded for non-ribosomal peptide synthase, NRPS) was too complex for IDT to manufacture (the manufacturing complexity score was 7 when checked with IDT’s gblocks tool, https://eu.idtdna.com/site/order/gblockentry). This difficulty score would have made our gene difficult to manufacture, and would have subsequently increased the length of time taken for this gene product to arrive to our lab. The manufacturing difficulty turned out to be because a stretch of the DNA sequence (starting at base 1111) had too many guanine bases next to each other.

To avoid this difficulty in manufacturing our gene product, the sequence of the gene was changed from GGG GGG GTG GGC G to TTA GGC GGT TGG GCC. The resulting sequence was also codon optimised to account for the abundance of nucleotides in our E. coli chassis. The resulting change was re-checked with the IDT gblocks tool, and the difficulty score had been reduced enough that we were able to use this new sequence in our order.  All the genes (coding for ATPG, NRPS, DHQS, and O-MT) were then checked to have the ATG start codon (which is needed in E. coli).  

This new part for NRPS is an updated CDS of the codon optimised NRPS from our Phase 1 project (Part:BBa_K3634003), which now has a slightly changed sequence to accommodate manufacturer (IDT) specifications. It’s new biobrick number is Part:BBa_K393000. NRPS is the final constitutive gene in the Shinogen cluster which converts sedoheptulose 7-phosphate to shinorine. The product, shinorine, has UV protectant properties as categorised by the Minnesota 2012 iGEM team (BBa_K814003). The gene was taken from cyanobacteria species Anabaena varThanogen – the Kill-switch: Our St Andrews Phase II team tried confirm whether the Thanogen parts (BBa_K3634006 to BBa_K3634021) designed by the 2020 (Phase I) St Andrews iGEM were fit for manufacture during the middle of summer. It was discovered that many of these parts were actually not fit for manufacturing by IDT – hence they could not be ordered and laboratory testing could not be carried out on the kill-switch during this 2021 phase of our Shinescreen project. These thanogen parts (linked below) had extremely high manufacturing complexity scores according to the IDT gblocks website (for example, one of the total complexity scores came to 340.9, whereas IDT identifies scores of 10 or greater to be denied for manufacture as the complexity is too high). Due to this extreme difficulty in manufacture, and limited time on our part to carry out laboratory research, our team decided to dedicate our time in the lab to implementing and testing the Shinogen construct, rather than rectifying the complexity of these sequences (or designing them anew). Improving the Thanogen parts to optimize them for manufacture (or re-designign them) is something that could be done in the future (i.e. during future phases of the Shinescreen project) for later testing and development of the gene circuit.  iabilis (ATCC 29413) and optimised for E. coli by our 2020 team. This year, it has been further corrected for IDT manufacturer. 

  Due to this change, NRPS was able to be manufactured, and therefore we have contributed to the registry with a part that is ‘ready to go’ and can be utilised by future teams looking to conduct research into the Shinogen pathway or other pathways that utilise this gene. Although this part is now fit to use, it could be further improved in the future by the re-addition of the composite part (with ATPG) to create a constitutive gene block with both sections of the gene pathway included (something our 2020 team discuss in silico, but this would need to be achieved in the lab for an improvement). The link to the registry can be found here

Hence, the Biobrick parts we used for Shinogen this year were: ATPG

NRPS

DHQS

0-MT

Thanogen-the kill switch

Our St Andrews Phase II team tried confirm whether the Thanogen parts (BBa_K3634006 to BBa_K3634021) designed by the 2020 (Phase I) St Andrews iGEM were fit for manufacture during the middle of summer. It was discovered that many of these parts were actually not fit for manufacturing by IDT – hence they could not be ordered and laboratory testing could not be carried out on the kill-switch during this 2021 phase of our Shinescreen project. These thanogen parts (linked below) had extremely high manufacturing complexity scores according to the IDT gblocks website (for example, one of the total complexity scores came to 340.9, whereas IDT identifies scores of 10 or greater to be denied for manufacture as the complexity is too high). Due to this extreme difficulty in manufacture, and limited time on our part to carry out laboratory research, our team decided to dedicate our time in the lab to implementing and testing the Shinogen construct, rather than rectifying the complexity of these sequences (or designing them anew). Improving the Thanogen parts to optimize them for manufacture (or re-designign them) is something that could be done in the future (i.e. during future phases of the Shinescreen project) for later testing and development of the gene circuit. 

ccaS

  ho1

  pcyA 

  ccaS/ho1/pcyA Composite Part

LacO- and LacP

  ccdB 

  Glucose Mediated Death Sensor

  CcdAB-Controlled mf-Lon Protease

  Lac and mf-Lon Degradation Tag

  Placlq and Lac Repressor (with mf-Lon Degradation Tag)

    ccaR

  PL8-UV5

  ccaR-Mediated ccdA Expression System

  CviJI Endonuclease (with ssrA degradation tag)

  LacI-Controlled CviJI Endonuclease (with ssrA degradation tag)