A protein array containing antigens specific for different diseases is one of the main parts of the DiaCHIP. Manufacturing of this array can be done by conventional protein expression and spotting of the proteins on a specific surface by hand or via cellfree expression. Our project aimed on establishing a cellfree expression system for expression of the antigens. Nevertheless, we made use of different Escherichia coli strains for overexpression of the antigens. Thereby we could start to establish the detection method while we were still working on optimizing our cell-free expression system. Both systems are based on different expression backbones. Therefore, a lot of cloning was required to obtain all constructs.
To reduce this ambitious task to a minimum of effort, we elaborated a well-structured cloning strategy inculding a self-designed multiple cloning site. The combination of classical cloning approaches and Gibson Assembly enabled a standardized cloning procedure that could easily be followed by many people working together in parallel. The cloning site for these purposes was incorporated into the commercial expression vector pET22b+ resulting in pET_iGEM.
We soon realized that protein expression is probably a problem many iGEM teams around the world are facing during their projects. Therefore, we decided to improve the plasmid backbone pSB6A1 for protein overexpression providing the registry with pOP, an expression backbone suitable for iGEM standard cloning procedures.
The establishment of our cloning strategy required a deep discussion about various cloning methods to find out about their advantages. 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.

1. Our own cloning site

2. Design of pET_iGEM

3. Detailed cloning strategy

4. pOP - Protein expression meets iGEM standards

5. Short review on cloning methods

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 is. A first calculation, based on the assumption of using about 15 antigens and 4 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 point is not possible¹⁾. To be able to exchange tags at the N-terminus as well as at the C-terminus, distinct restriction sites need to remain between the parts. Moreover, we first wanted to establish an expression cassette inserted between BioBrick pre- and suffix of the submission backbone pSB1C3, so making use of an additional RFC was not possible.

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.

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 AlfII restriction sites (Fig. 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 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).

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].

Figure 3: pIG15_002. 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.

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 Figure 2.
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. 3).

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 Figure 4.
Using this expression vector exhibits a lot of advantages compared to working with a pSB1C3-based vector. One advantage is that the vector is already established for protein expression, so we could access the experience of more practised people. Additionally, this vector carries an ampicillin resistance as a selection marker instead of the chloramphenicol resistance in pSB1C3. This is advantageous in the case of protein expression because many E. coli strains, which are adapted for this purpose, are chloramphenicol-resistant. Thus, a double selection for successful transformation would not be possible.
However, the cell-free expression backbone was not changed because the template for expression is a PCR product amplified from promoter to terminator. This means the backbone, which is used for cloning the construct, is irrelevant for protein expression. So, we decided to clone the constructs in a cloning-optimized, high copy plasmid, pSB1C3.

Figure 4: Comparison of pET_iGEM and pET22b+. 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.

As mentioned before, we established a cloning strategy which is easy to handle for a lot of people. All our cloning efforts begin 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+ is inserted into pSB1C3.

The basic constructs (protein purification using E. coli)

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 extention.
pET_803: To insert the very small but commonly used His-tag, we again used Gibson Assembly, because classical cloning of such small parts is hard. The respective primers were designed for amplification of antigen 8 (Herpes simplex TypeI, glycoprotein G1) to combine the insertion of the tag and an antigen. While the forward primer was a common 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_804: Another tag we use for immobilization of the antigens on the surface is the Spy-tag. 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 is needed for protein purification. Consistent with our cloning strategy, the amplified part was inserted between Acc65I and AflII, because it contains an N-terminal as well a a C-terminal tag. BamHI and HindIII restriction sites were retained flanking the antigen making it exchangable.
pET_805: The only tag we used which was not inserted by Gibson Assembly was the Halo-tag. With a length of about 900 bp, classical cloning of this part was no trouble. The Halo-tag exists in two variants, one being optimized for C-terminal tagging of the protein, the other for N-terminal tagging (Ref. Promega?). As the tag we have access to is the C-terminal one, the Spy-tag in pET_804 was exchanged using the restriction sites HindIII and AflII resulting in pET_805.

Tagged constructs (protein purification using E. coli)

We wanted to establish a tag-system which minimizes unspecific binding of proteins to the chip surface, therefore we tried out different tag systems.
His-tag: Every antigen was genetically fused to a C-terminal 10xHis-tag, which could be used for its immobilization on the surface as well es for its purification. The basic construct for this purpose was pET_803. The Herpes simplex derived antigen which was initially completed had to be excised by restriction with BamHI and HindIII. Thus, all the other antigens digested in the same way could be ligated into this backbone.
Spy-tag: Another tag for surface immobilization of the antigen is the Spy-tag. To enable protein purification via Ni-NTA constructs with Spy-tag were additionally fused to a C-terminal 6xHis-tag. The basic vector for cloning of those constructs was pET_804. Excision of the Herpes simplex derived antigen and insertion of other antigens was done analogous to antigen exchange in His-tag constructs.
Halo-tag:As a second covalent tag-system for immobilization of the antigens to the surface we used the Halo-tag, which specifically binds to a chloroalkane surface. Using our own cloning strategy, the Halo-tag can be easily inserted between HindIII and AflII, using pET_803 as basis. Cloning of the Halo-tag into pET_803 leads to a new vector, pET_805. This vector is then used to substitute the antigens. As in the Spy-tag-construct, this new vector contains a N-terminal 6x His-tag for purification of the antigens.
HA-tag:?

Cellfree backbone

As described above we decided to use the pSB1C3 standard vector as backbone for our cellfree expression constructs. Inserted between the RFC 10 prefix and suffix is our own cloning site, containing the restriction sites: Acc65I, BamHI, HindIII and AflII. We included a T7 promotor and a ribosomal binding site between the RFC 10 prefix and the Acc65I restriction site. A Stopcodon-cassette and a T7 terminator were inserted between the AflII restriction site and the RFC 10 suffix. These similarities to the expression vector facilitate the easy exchange of fragments between the expression vector and the cell free vector. Using our own cloning site it is rather simple to exchange different tag-combinations. The advantage of the pSB1C3 backbone is the high copy origin, allowing a rapid multiplication and easy cloning. For the cellfree expression itself the vector backbone is dispensable as only the PCR amplified part between the prefix and the suffix is mandatory for cellfree expression.

The basic constructs (cell free expression)

pIG15_104: For the first basic construct we ordered a gBlock from IDT containing T7 promotor, ribosomal binding site, our cloning site and the T7 terminator with adjoining Stopcodon-cassette. Between BamHI and HindIII the coding sequence for tYFP was inserted and between HindIII and AflII the Spy-tag sequence. This gBlock was then inserted into the pJET vector. Afterwards it was digested out of pJET and classically cloned into the pSB1C3 backbone resulting in the first basic construct for cell free expression. We inserted tYFP as default fragment into the cellfree expression backbone because we already had some anti-tYFP available. Hence first measurements for analysis of the binding of anti-TYFP to tYFP could be made as soon as the basic construct was finished. Besides tYFP could be used as reporter. Using BamHI and HindIII restriction sites tYFP can be excised and replaced by antigen sequences analogous to the cloning system used for the expression vector.
pIG15_105: Using the HindIII and AflII restriction sites the Spy-tag sequence was excised. For our second covalent tag system we inserted the sequence for the Halo-tag via classical cloning thereby producing our next basic construct for cellfree expression. Using identical cloning strategies as with the first basic construct (pIG15_104) all antigen sequences can be inserted into this vector using BamHI and HindIII.