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<h1 class="sectionedit1">Cloning site</h1>
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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 <em>E. coli</em> 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. 
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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 <strong>both</strong> 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 <acronym title="Request for Comments">RFC</acronym>[10], so using another iGEM standard between pre- and suffix is not possible.
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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. <br/>

Finally, this resulted in a cloning site containing Acc65I-, BamHI-, HindIII- and AlfII restriction sites (<strong>figure x</strong>). 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. <br/>

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.
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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).
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<h1 class="sectionedit2">Vector Design</h1>
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As all our constructs are supposed to be overexpressed and purified either in <em>E. coli</em> or via cell-free expression, we wanted to combine the advantages of the commonly used expression vector <a class="media mediafile mf_pdf" href="/igem2015/lib/exe/fetch.php?media=labjournal:cloning:pet22b.pdf" title="labjournal:cloning:pet22b.pdf">pET22b(+)</a> with the iGEM standard vector  <a class="media mediafile mf_pdf" href="/igem2015/lib/exe/fetch.php?media=labjournal:cloning:psb1c3.pdf" title="labjournal:cloning:psb1c3.pdf">pSB1C3</a> which allows us to hand in all our constructs to the iGEM registry. <br/>

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 <strong>Fig. 001</strong>.<br/>
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<div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/a/a1/Freiburg_labjournal-cloning-pig15_001neu.jpg" title="labjournal:cloning:pig15_001neu.jpg"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/a/a1/Freiburg_labjournal-cloning-pig15_001neu.jpg" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/a/a1/Freiburg_labjournal-cloning-pig15_001neu.jpg" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Fig. 001</div></div></div><hr/>
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The backbone for <em>E. coli</em>-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 (<strong>Fig. 002</strong>).
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<div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/a/ac/Freiburg_labjournal-cloning-pig15_002neu.jpg" title="labjournal:cloning:pig15_002neu.jpg"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/a/ac/Freiburg_labjournal-cloning-pig15_002neu.jpg" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/a/ac/Freiburg_labjournal-cloning-pig15_002neu.jpg" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Fig. 002</div></div></div><hr/>
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Unfortunately, after the first construct was ready for expression in <em>E. coli</em>, 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 <strong>figure x</strong>. <br/>
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<div class="thumb2 trien" style="width:910px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/3/37/Freiburg_labjournal-cloning-petcompare2neu.png" title="labjournal:cloning:petcompare2neu.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/3/37/Freiburg_labjournal-cloning-petcompare2neu.png" width="900"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/3/37/Freiburg_labjournal-cloning-petcompare2neu.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div></div></div></div><hr/>
<div class="thumb2 trien" style="width:910px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/1/15/Freiburg_labjournal-cloning-petcomp_wiki2.jpg" title="labjournal:cloning:petcomp_wiki2.jpg"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/1/15/Freiburg_labjournal-cloning-petcomp_wiki2.jpg" width="900"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/1/15/Freiburg_labjournal-cloning-petcomp_wiki2.jpg" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div></div></div></div><hr/>
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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 <em>E. coli</em> strains, which are adapted for this purpose, are chloramphenicol-resistant. Thus, a double selection for successful transformation would not be possible. <br/>

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.
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