One very basic task of our project is the cloning of all constructs that enable the expression of every antigen with several different tags. Additionally, those constructs have to be adapted for standard overexpression in E. coli as well as for cell-free expression. A first calculation, based on the assumption of using about 15 different proteins and 4 different tags, resulted in a total of 120 constructs to be cloned. Therefore, we decided to create our own multiple cloning site which could be used to combine every antigen with the needed tag(s) in a standardized matter, making it easy for many different people to work with it.

As a new cloning site, we did not use one of the available iGEM standards for several reasons. First, we need at least three distinct sites which can be used for insertion of a part, one for an N-terminal tag, one for an antigen and one for a C-terminal tag. Using, for example, SpeI and XbaI sites to insert a part in the middle would result in a 50 % chance to have the insert cloned in the wrong orientation, because SpeI and XbaI restriction both produce the compatible overhang GATC. Another reason is that we originally planned to insert the whole expression cassette into pSB1C3. This iGEM standard plasmid already contains RFC[10], so using another iGEM standard between pre- and suffix is not possible.

The basic idea was to have distinct restriction sites which can be used for the insertion of an antigen, while others can be used for the exchange of tags.
Finally, this resulted in a cloning site containing Acc65I-, BamHI-, HindIII- and AlfII restriction sites (figure x). Using those sites for the assembly of different antigen-tag combinations will never result in a frameshift, if the coding sequence is in frame with the restriction sites flanking it.

Fig. 001: Our own cloning site for containing Acc65I, BamHI, HindIII and AflII,that are assembled in a way that facilitates exchange of tags without getting a frameshift.

In practice, this means that the restriction sites BamHI and HindIII can be used to insert an antigen by classical cloning. Afterwards, either Acc65I and BamHI can be used to add an N-terminal tag or HindIII and AflII can be used to fuse a C-terminal tag to the antigen. Linker regions, creating some space between the tag and the antigen to avoid interactions between them, should be attached to the tag sequence. Other combinations of restriction sites can be used to insert antigen-tag combinations which might already be available. In addition to classical ligation, the respective restriction sites can be used to open the backbone for other cloning methods, e.g. Gibson Assembly.

The restriction sites have been chosen according to the amino acids which result by translation of their sequence. Acc65I (G/GTACC) and BamHI (G/GATCC) are translated into glycine and either serine or threonine. Those are hydrophilic, soluble amino acids which are commonly used in flexible linkers. The translation of HindIII (A/AGCTT) results in lysine and leucin. Especially lysine is highly hydrophobic, which is why some soluble amino acids should be used as a linker in front of a C-terminal tag. The last restriction site, AflII (C/TTAAG), is also translated to leucin and lysine. This will not much affect protein function or folding since these are the last translated codons. But in order to prevent rapid protein degradation according to the N-end-rule in bacteria, few additional amino acids have been inserted between the AflII site and the stop codon (TGA).

As all our constructs are supposed to be overexpressed and purified either in E. coli or via cell-free expression, we wanted to combine the advantages of the commonly used expression vector pET22b(+) with the iGEM standard vector pSB1C3 which allows us to hand in all our constructs to the iGEM registry.
Therefore, the main features of pET22b(+) were integrated between the BioBrick prefix and suffix of pSB1C3. Those features are namely the T7 promoter and terminator, a ribosomal binding site, the lacI gene and a signal sequence for periplasmic translocation of the protein (pelB). In between the pelB sequence and the terminator, a cloning site is arranged which we designed to simplify the cloning intents (see next section for more details). The resulting plasmid pIG15_001 is shown in Fig. 002.

Fig. 002: pIG15_001 contains the pSB1C3 backbone with an altered cloning site for improved assembly of protein coding sequences with different tags (N- or C-terminal), which avoids the formation of frameshifts. Important parts for overexpression of proteins in E. coli were added inside the RFC[10].

The backbone for E. coli-based cell-free expression purposes was designed analogous, but without a pelB secretion signal and the lacI gene, because those are not needed for cell-free expression (Fig. 003).

Fig. 003: For cell-free expression the pSB1C3 backbone was altered similarly, but without a pelB secretion signal or lacI coding sequence and with a CMV promotor instead of a T7 promoter.

Unfortunately, after the first construct was ready for expression in E. coli, we realized that it did not work as expected. Instead of wasting time trying to optimize our own expression vector, we went on using the original vector and modified the cloning site to fit our requirements. The original vector pET22b+ and the modified version pET_iGEM are compared in (Fig. 004).

Fig. 004: The two vectors differ in the the cloning site, which contains only four restriction sites in pET_igem (Acc65I, BamHI, HindIII, AflII) compared to the more complex multiple cloning site of the original pET22b+ (BamHI, EcoRI, SacI, SalI, HindIII, NotI, AvaI, XhoI). Also the 6x His-Tag from pET22b+ was removed, to enable easy tag-exchanges with our own system.