Team:Hong Kong HKU/plasmidesign

Plasmid Design

The primary objective of constructing the plasmid pETFlexStack was to allow users to clone in any Biobricks compatible with the RFC10 standards, using just one or even no pair of universal primer, with flexible alterations that is as simple as possible.

For the features:
1.    Any biobrick, in any standard plasmids, with a start codon immediately downstream to the Standard Prefix, can be cloned and fused with the signal peptide using XbaI and PstI digestion of the Biobrick-carrying plasmid, and ligated to pETFlexStack linearized by SpeI and PstI. The linker so-formed by this ligation is precisely designed to encode for a hydrophilic, flexible peptide of threonine, glycine, and serine.
2.    As the standard Prefix and Suffix system is used, users can of course restrict the whole signal peptide-fused biobrick out into the pSB1C3 or other iGEM plasmids for cloning and gene assembly purposes.
3.    Benefiting from the design of the Prefix and Suffix system, the rapid stacking assembly of genes can also be done on pETFlexStack. To facilitate convenient expression of the signal peptide-fused cargo proteins, a T7 RBS is also included within the Prefix-Suffix region, so that even though the genes are stacked they would still retain their own RBS for expression.
4.    Sometimes if the users would like to have individual promoters for individual signal peptide-fused genes, the gene stacking can still be performed, made possible by a NheI site just upstream to the T7 promoter controlling the transcription of the cargo protein gene. This design took advantage of the fact that NheI, SpeI and XbaI are all compatible with each other and commonly present in most cloning laboratories.
5.    The individual promoters and RBS governing the expression of a cargo gene can be changed using their flanking restriction sites provided, however, this has to be done prior to gene stacking, after which there would be multiple digestion sites for some restriction sites. This would be an aspect that deserves improvements to make such alteration even simpler and more convenient.
6.    SacI and BamHI restriction sites are located in close proximity to the start codon of EutS, which allows one to fuse affinity and recognition tags to EutS, the richest protein in composition in Eut BMC. The SacI site was chosen and arranged to code for a hydrophilic and flexible linker S-S-G-S after cloning. After affinity fusion, the spacing between the RBS and the start codon would be precisely 8 bp, the optimal for ribosomal translation.
7.    Since previous works on the Eut BMC revealed that EutS, without the other components (EutM, EutN, EutL, EutK), can form a pseudo-Eut BMC that is comparable in morphology and size to the native Eut BMC, we strategically added a BglII site that, if pETFlexStack is digested with BglII and subsequently self-ligated (in intervening sequence is digested into 2 pieces), the genes coding for EutM, EutN, EutL and EutK could be deleted, leaving the tagged EutS behind under its promoter. This would allow easy construction of genetic systems to compare the EutS BMC with the native BMC.
8.    If one would like to segregate EutSMNLK and the cargo-coding gene in two plasmids, or for experimental control purposes, the entire EutSMNLK gene region can be removed by double digestion with BglII and BamHI, and subsequently ligation would give a plasmid that codes only for the cargo proteins. This is possible due to the compatible ends of BglII and BamHI digestion products.