A protein array containing antigens from different diseases is one of the main parts of the DiaCHIP. Manufacturing of this array can be done by "traditional" protein expression and spotting of the proteins on a specific surface or via cellfree expression. Our project focused on establishing a cellfree expression system for expression of the antigens but nevertheless we used different E. coli strains for expression of proteins as backup. Working with this two systems in parallel, cellfree expression on the one hand and expression with E. coli on the other hand, involved a lot of cloning.
That is why we designed our own cloning strategy and multiple cloning site which are adapted to our project. After some initial setbacks we finally found the system best suited for our needs, a vector we called pET_igem.
We soon realised that protein expression is probably a problem many iGEM teams around the world are facing during their projects. Therefore we decided to improve the original pSB1C3 in a similar way, thus providing an iGEM conform backbone (pOP)that can be used by other teams for easy expression of proteins.
During our cloning work we mainly used two methods: Classical cloning using restriction enzymes and Gibson cloning. We really got to appreciate the differences and advantages of the two different methods, which inspired us to write a short review comparing the two systems.

1. Our own cloning site

2. Vector design

3. Classical cloning vs. Gibson cloning

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 (Fig. 001). 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.

Figure 1: Self-designed cloning site: Our own cloning site containing the restriction sites 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).