Assembly of the stf gene
For the assembly of the bacteriophage Lambda tail fibre protein (stf gene), our initial step was the design of ORF314. This sequence was optimised to remove any illegal restriction sites before having the entire gene synthesised. This sequence was initially cloned into both the linearised shipping plasmid pSB1C3 to create our first BioBrick (see details here) and into the linearised assembly plasmid pSB1K3 for further processing, using the standard iGEM restriction protocol with EcoRI and PstI enzymes, followed by ligation using T4 DNA ligase as per the standard iGEM protocol. Following purification of the plasmids using the Thermo Scientific GeneJET plasmid miniprep kit, the part size was confirmed by setting up a 10 μL restriction with EcoRI and PstI, followed by agarose gel electrophoresis (15 W, 85 mA, 150 V (limiting factor)) (Fig 1).
Fig 1. Agarose gel electrophoresis to confirm presence of BioBrick in shipping plasmid.
A 6x His-tag was subsequently added to the construct within the assembly backbone (Fig. 2) by carrying out site-directed mutagenesis to introduce restriction sites for the Type 2 restriction enzyme BsaI before the stop codon of the ORF314 sequence (Fig. 3). The construct was then restricted with this enzyme before being annealed and ligated with a pair of oligonucleotides with compatible overhangs, which contained the His-tag sequence replacement BioBrick suffix sequences.
Fig 2. Synthesised oligonucleotides containing a His-tag and BioBrick suffix.
Fig 3. Construct containing Assembly plasmid pSB1K3 and ORF314 (a) showing location of primers creating BsaI cut sites, and (b) after restriction-ligation.
After addition of the His-tag, both the recombinant pSB1K3 plasmid and our synthesised ORF401 gene were restricted with the blunt-end restriction enzyme EcoRV and the sticky-end enzyme EcoRI as per the standard iGEM protocol, in order to create incompatible ends. The two products were combined and ligated with T4 DNA ligase – again according to standard iGEM protocol – before agarose gel electrophoresis was carried out to determine the success of the restriction-ligation. Results can be viewed here.
To create the complete stf gene, we will now carry out 3A assembly. We will restrict the above construct with EcoRV and PstI restriction enzymes to remove the ORF314 gene including the 6x His-tag. Consecutively, we will restrict the linearised backbone pSB1C3 with EcoRI and PstI and the previously synthesised sequence ORF401 will be restricted with EcoRI and EcoRV. As such, we will create three fragments that can only be combined in a single combination, which we will anneal and ligate using T4 DNA ligase as per the approved iGEM protocol. We will then transform these recombinant plasmids into 10β competent cells and carry out antibiotic selection to exclude any recombinants containing the pSB1K3 backbone. Successful colonies will be grown in overnight cultures, before a plasmid miniprep (Thermo Scientific GeneJET) will be carried out to purify the plasmids. The correctness of the construct will be confirmed both by carrying out single and double restriction of the plasmid before agarose gel electrophoresis, and through Sanger sequencing. On confirmation of the sequence, the construct will be submitted as a BioBrick.
Additionally, the stf construct will be cloned into a previously generated vector containing a promoter, ribosome binding site and terminator to facilitate characterisation of the part, as well as to create a new composite part.