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Revision as of 19:42, 18 September 2015
DNA Engineering
A protein array containing different antigens specific for distinct diseases is one of the main parts of the DiaCHIP. Manufacturing this array can be done by conventional protein expression and spotting of the proteins on a specific surface by hand or via cell-free expression. 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 including a self-designed multiple cloning site. 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.
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 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.
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).
2. 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.
The vector for expression in mammalian cells(pIG15_002; figure 3) 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.
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 making the vector it is assembled on irrelevant. Using a high-copy plasmid as pSB1C3 rather simplifies cloning efforts.
3. Detailed Cloning Strategy
As mentioned before, we established a cloning strategy, which 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)
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_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_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.
pET_805: The only tag we used that was not inserted via Gibson Assembly was the HaloTag. With a length of about 900 bp, classical cloning of this part was no trouble. The HaloTag exists in two variants, one being optimized for C-terminal tagging of the protein, the other for N-terminal tagging. As the tag we had access to was the C-terminal one, the SpyTag 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 testing 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 as for its purification. The basic construct for this purpose was pET_803. The Herpes Simplex Virus derived antigen, which was initially finished cloning, 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.
SpyTag: Another tag for surface immobilization of the antigen is the SpyTag. To enable protein purification via Ni-NTA columns proteins fused to the SpyTag were additionally fused to a C-terminal 6xHis-tag. The basic vector for cloning these constructs was pET_804. Excision of the Herpes Simplex Virus derived antigen and insertion of other antigens was performed analogous to the antigen exchange in His-tag constructs.
HaloTag: As a second covalent tag-system for immobilization of the antigens on the surface we used the HaloTag, which specifically binds to a chloroalkane surface. Using our own cloning strategy, the HaloTag could easily be inserted between HindIII and AflII, using pET_803 as basis. Cloning of the HaloTag into pET_803 leads to a new vector, pET_805. This vector is then used to substitute the antigens. As in the SpyTag construct, this new vector contains a C-terminal 6xHis-tag for purification of the antigens.
Cell-Free Backbone
As described above we decided to use the pSB1C3 standard vector as backbone for our cell-free expression constructs. Inserted between the RFC 10 prefix and suffix is our own cloning site, containing the following restriction sites: Acc65I, BamHI, HindIII and AflII. We included a T7 promoter and a ribosomal binding site between the RFC 10 prefix and the Acc65I restriction site. A stop codon 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 standard expression vector and the cell-free expression vector. Using our 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 cell-free expression itself the vector backbone is dispensable as only the part amplified via PCR between the prefix and the suffix is mandatory for cell-free expression.
The Basic Constructs (Cell-Free Expression)
pIG15_104: For the first basic construct we ordered a gBlock from IDT containing the T7 promoter, ribosomal binding site, our cloning site and the T7 terminator with adjoining stop codon cassette. Between BamHI and HindIII the coding sequence for tYFP and between HindIII and AflII the SpyTag sequence was inserted. This gBlock was then inserted into the pJET1.2 vector. Afterwards, it was digested out of pJET1.2 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 cell-free expression backbone because we already had some anti-tYFP antibody available. Hence, first measurements for analyzing the binding of anti-tYFP to tYFP could be performed as soon as the basic construct was finished. Besides, tYFP could be used as reporter. Using BamHI and HindIII restriction sites tYFP could 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 SpyTag sequence was excised from pIG15_104. For our second covalent tag system we inserted the sequence for the HaloTag via classical cloning and thereby producing our next basic construct for cell-free expression, pIG15_105. This backbone again contains tYFP as default insert, this time with a C-terminal HaloTag.
Using identical cloning strategies as with the first basic construct (pIG15_104) all antigen sequences could be inserted into this vector using BamHI and HindIII.
