DNA Engineering

A protein array containing different antigens specific for distinct diseases is one of the main parts of the DiaCHIP. Obtaining all the DNA constructs requires a lot of cloning, especially because conventional protein expression and expression using a cell-free system are based on different plasmid backbones.
To reduce this ambitious task to a minimum of effort, we elaborated a well-structured cloning strategy including a self-designed multiple cloning site. This site was incorporated into the commercial expression vector pET22b+ resulting in pET_iGEM.
We soon realized that efficient protein expression is a problem many iGEM Teams around the world may be facing during their projects. Therefore, we provide the Registry with pOP, an expression vector suitable for iGEM standard cloning procedures.
To help future iGEM Teams with their decision, which cloning method to use, we compared and contrasted classical cloning and Gibson Assembly in a short review.

Our Cloning Site

The cloning site we developed enables the assembly of different antigens with a number of tags in a standardized manner making it simple for many people to work with. A first calculation based on the assumption of using about 15 antigens and four different tag systems resulted in a total of 120 constructs to be cloned. This underlines the need for a simplified cloning procedure.

We did not use one of the available iGEM standards as a new cloning site for several reasons.

The RFCs are designed to enable serial assembly of various sequences, but the exchange of single parts at a later time point is not possible¹⁾. In order to be able to exchange tags at the N-terminus as well as at the C-terminus, distinct restriction sites need to persist between the parts. Moreover, we first wanted to establish an expression cassette inserted between BioBrick pre- and suffix of the submission backbone pSB1C3. Therefore, making use of an additional RFC was not possible.

Figure 1: Self-designed cloning site. Our cloning site contains the restriction sites Acc65I, BamHI, HindIII and AflII, that are assembled in a way that facilitates exchange of tags without getting a frame-shift.

The basic idea was to have distinct restriction sites which can be used for the insertion of antigens, while others can be used for the exchange of tags. Finally, this resulted in a cloning site containing Acc65I, BamHI, HindIII and AflII restriction sites (figure 1). 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.
The restriction sites have been chosen according to the amino acids that result from the 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 highly affect protein function or folding since these are the last translated codons. Anyways, in order to prevent rapid protein degradation according to the N-end-rule in bacteria²⁾, a few additional amino acids have been inserted between the AflII site and the stop codon (TGA).

¹⁾Knight T 2003. Idempotent Vector Design for Standard Assembly of BioBricks.

²⁾Tobias JW 1991. The N-end rule in bacteria. Science.

Design of pET_iGEM

Figure 2: pIG15_001. pSB1C3 backbone containing an altered cloning site in the middle of an expression cassette 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].

For protein expression, we designed two vectors based on the submission backbone pSB1C3. pIG15_001 (figure 2) was supposed to be adapted for inducible protein overexpression in E. coli. Therefore, we inserted main features derived from the commercial expression vector pET22b+ between the BioBrick pre- and suffix of pSB1C3. These include a T7 promoter and terminator, a ribosomal binding site, the self-designed cloning site and a lacI expression cassette. Additionally, a pelB signal sequence for periplasmic translocation was added 5’ to the cloning site.

Figure 3: pIG15_104. For cell-free expression the pSB1C3 backbone was altered similarly, but without a pelB secretion signal or lacI coding sequence and with a CMV promoter instead of a T7 promoter. In this case, the coding sequence for TurboYFP is inserted.

The vector for cell-free expression (pIG15_104) was designed analogously, but lacking the lacI expression cassette and the signal sequence. The T7 promoter and terminator were exchanged by a CMV promoter and a WPRE terminator region for secretion into the medium. In figure 3, the vector is shown with an inserted coding sequence for TurboYFP.

Unfortunately, after the first expression experiment in E. coli, we realized that the yields of protein expression are not sufficient for our purposes. 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 figure 4.

Using an adapted commercial expression vector finally resulted in sufficiently high protein yields. The advantages of expression vectors like pET22b+ in contrast to cloning vectors like pSB1C3 are discussed in detail on the introduction page for pOP. This is a plasmid backbone we submitted to the iGEM Registry that is optimized for protein overexpression purposes and fully compatible with iGEM idempotent cloning standards of RFC[25].
The backbone for cell-free expression was not changed because a PCR product of the expression cassette serves as a template for the actual expression purpose. Therefore, the vector that was used for its assembly is irrelevant. Using a high-copy plasmid such as pSB1C3 rather simplifies cloning efforts.

Figure 4: Comparison of pET_iGEM and pET22b+. The two vectors differ in the cloning site that 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). The 6xHis-Tag from pET22b+ was also removed to enable easy tag-exchanges with our system.

Detailed Cloning Strategy

As mentioned before, we established a cloning strategy that is easy to handle for a team of researchers working together on the same project. It facilitates easy exchange of tags for custom purposes. All our cloning efforts began with either pET_iGEM (the modified version of pET22b+) for E. coli-based protein expression or pIG15_104, where the expression site derived from pET22b+ was inserted into pSB1C3.

The Basic Constructs (Protein Purification Using E. coli)

pET22b+ with our cloning site (pET_iGEM)

To modify the multiple cloning site of pET22b+ for our purposes, we performed a Gibson Assembly with only one fragment. Therefore, we designed primers for the amplification of the whole plasmid, except the MCS. The cloning site we designed ourselves was part of the primer extension.

pET_iGEM with His-tagged Herpes simplex antigen (pET_803)

To insert the very small but commonly used His-tag, we again used Gibson Assembly because classical cloning of such small parts can be a challenging task. The respective primers were designed for amplification of antigen 8 (Herpes Simplex Virus Type 1, glycoprotein G1) to combine the insertion of the tag and an antigen. While the forward primer was a regular Gibson primer, the reverse primer additionally contained the sequence of a 10xHis-tag. After amplification, the part was inserted into the BamHI and AflII digested backbone pET_iGEM resulting in the first antigen with N-terminal His-tag.

pET_iGEM with Spy-tagged Herpes simplex antigen (pET_804)

Another tag we wanted to use for immobilization of the antigens on the surface is the SpyTag. This part of 39 bp length was inserted into pET_iGEM analogous to the 10xHis-tag. The only difference was that the forward primer for amplification of antigen 8 included a standard 6xHis-tag, which enables the purification of the protein. Consistent with our cloning strategy, the amplified part was inserted between Acc65I and AflII as it contains an N-terminal as well as a C-terminal tag. BamHI and HindIII restriction sites were retained flanking the antigen, making it freely exchangeable.

Team:Freiburg/Project/DNA Engineering