Team:USyd-Australia/pUS201

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<tr><td > <h7> <a href="http://parts.igem.org/Part:BBa_K1388000">pUS203</a></h7></td>
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<tr><td > <h2> <a href="#">pUS201 Low Copy Plasmid</a></h2></td>
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<h3>Aim</h3>
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<p>To produce a low copy plasmid backbone for the cloning of integron integrase (IntI1). The integron integrase (IntI1) is believed to be toxic to cells when expressed at high concentrations, thus, it was expected that it would benefit the stability of the integrase construct if it was both controllable, and expressed on a low copy plasmid backbone. </p>
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<li style="font-weight: bold;"> <h2 onclick="toggle_visibility('PlasmidMap');">Plasmid Map of pUS201</h2></li>
 
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<img src="https://static.igem.org/mediawiki/2014/6/67/PUS203.png" >
 
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<h3>Approach</h3>
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We sought to improve one existing low-moderate copy backbone, <a href=http://parts.igem.org/Part:pSB6A1>pSB6A1</a>, for this purpose. pSB6A1 is derived from pBR332, having a pMB1 ori site, a Rop gene that acts to reduce copy number and ampicillin resistance as the selectable marker. Additionally, pSB6A1 has had a biobrick prefix/suffix cloning site suffix cloning region added, which has disrupted the tetracycline resistance gene present in pBR332. <br><br>
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The integron integrase (IntI1) is believed to be toxic to cells when expressed at high concentrations, thus, it was expected that it would benefit the stability of the integrase construct if it was both controllable, and expressed on a low copy plasmid backbone. We sought to improve one existing low-moderate copy backbone, <a href=http://parts.igem.org/Part:pSB6A1>pSB6A1</a>, for this purpose. pSB6A1 is derived from pBR332, having a pMB1 ori site, a Rop gene that acts to reduce copy number and ampicillin resistance as the selectable marker. Additionally, pSB6A1 has had a biobrick prefix/suffix cloning site suffix cloning region added, which has disrupted the tetracycline resistance gene present in pBR332. <br><br>
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A large region of defective TetR gene sequence is still remaining, and the cloning region lacks terminators either side of the prefix and suffix. Thus, we sought to assemble a new plasmid, pUS201, incorporating the AmpR, Ori and rop sites from pSB6A1, and the biobrick prefix/suffix cloning region with terminators from pSB1C3.<br>
A large region of defective TetR gene sequence is still remaining, and the cloning region lacks terminators either side of the prefix and suffix. Thus, we sought to assemble a new plasmid, pUS201, incorporating the AmpR, Ori and rop sites from pSB6A1, and the biobrick prefix/suffix cloning region with terminators from pSB1C3.<br>
<img src="https://static.igem.org/mediawiki/2014/5/5c/USYD2014_pUS201strategy.PNG" width="90%">
<img src="https://static.igem.org/mediawiki/2014/5/5c/USYD2014_pUS201strategy.PNG" width="90%">
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<p><b>Figure 1</b> – General strategy behind the construction of pUS201.<p>
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<p style="font-size:12px"><b>Figure 1</b> – General strategy behind the construction of pUS201.<p>
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<h3>Materials</h3>
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<li>DNA
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<ul><li><a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>: Used to PCR out the pSB6A1 backbone</li>
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PCR of the pSB6A1 backbone (from pSB6A1 containing <a href = http://parts.igem.org/Part:BBa_J04450 > BBa_J04450 </a> was done using <a href = https://2014.igem.org/Team:USyd-Australia/Notebook/Primers > primers </a> 1401 and 1402. Similarly, PCR of pSB1C3 (from pSB1C3 containing <a href = http://parts.igem.org/Part:BBa_K592009 > BBa_K59009 </a> yielded the biobrick cloning region. The overlap between the sequences of the PCR products allowed for Gibson assembly of the final plasmid, pUS201. This was transformed into Top10 E. coli, with the correct clone selected for on ampicillin, with a white colouration.
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<li><a href = http://parts.igem.org/Part:BBa_K592009 > BBa_K59009 </a>: Used a source of the BioBrick cloning region</li>
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<li>Primers
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<li><a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1401">iGEM1401</a>
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<li><a href="https://2014.igem.org/Team:USyd-Australia/Notebook/Primers#iGEM1402">iGEM1402</a>
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<p style="font-size:10px"><b>Figure 2</b> – Digestion of the plasmid preparation of pUS201 with a variety of restriction endonucleases yields the expected fragment sizes when visualised with agarose gel electrophoresis.  This suggests that the correct product had been constructed. </p>
 
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PCR of the pSB6A1 backbone (from pSB6A1 containing <a href = http://parts.igem.org/Part:BBa_J04450 > BBa_J04450 </a> was done using <a href = https://2014.igem.org/Team:USyd-Australia/Notebook/Primers > primers </a> 1401 and 1402. Similarly, PCR of pSB1C3 (from pSB1C3 containing <a href="http://parts.igem.org/Part:BBa_K592009"> BBa_K59009 </a> yielded the biobrick cloning region. The overlap between the sequences of the PCR products allowed for Gibson assembly of the final plasmid, pUS201. This was transformed into Top10 E. coli, with the correct clone selected for on ampicillin, with a white colouration.  
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We aimed to validate that pUS201 was low copy by observing the proliferation of pUS201 positive E. coli in different concentrations of ampicillin or carbenicillin. Unfortunately, the results indicated that pUS201 was not low copy, with the rate of proliferation of bacteria in solutions of LB and carbenicillin similar to pUC19, a high copy plasmid that expresses ampicillin resistance.<br>
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<img src="https://static.igem.org/mediawiki/2014/a/a7/USYD2014_pUS201geldigest.PNG" hspace="30px" height="40%" width="40%" align="right"><br>
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<img src="https://2014.igem.org/File:USYD2014_PUS201proliferationgraph.PNG" hspace="30px"><br>
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<p style="font-size:12px"><b>Figure 2</b> – Digestion of the plasmid preparation of pUS201 with a variety of restriction endonucleases yields the expected fragment sizes when visualised with agarose gel electrophoresis.  The results are as expected providing us with good evidence that the part is what we want. </p>
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<p style="font-size:10px"> Figure 3 – Proliferation of E. coli after 2.5 hours in LB media with various concentrations of carbenicillin, as measured by OD600. Error bars represent the standard error of the mean. pUS201 seems to behave similarly to pUC19, the high copy plasmid, as opposed to the low copy pSB6A1. </p>
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<h3>Validation</h3>
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We aimed to validate that pUS201 was low copy by observing the proliferation of pUS201 positive E. coli in different concentrations of ampicillin or carbenicillin. Unfortunately, the results indicated that pUS201 was not low copy, with the rate of proliferation of bacteria in solutions of LB and carbenicillin similar to pUC19, a high copy plasmid that expresses ampicillin resistance.<br>
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<img src="https://static.igem.org/mediawiki/2014/thumb/a/a8/USYD2014_PUS201proliferationgraph.PNG/800px-USYD2014_PUS201proliferationgraph.PNG" hspace="30px"><br>
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<p style="font-size:12px"> <b>Figure 3</b> – Proliferation of E. coli after 2.5 hours in LB media with various concentrations of carbenicillin, as measured by OD600. Error bars represent the standard error of the mean. pUS201 seems to behave similarly to pUC19, the high copy plasmid, as opposed to the low copy pSB6A1. </p>
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We made several attempts to validate this biobrick using 2 low copy backbones and finally just as it was. The low copyback  backbones were desired because Inti1 is toxic to the cells at high concentrations. <br><br>
 
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The first attempt was to put it into a low copy backbone we’d made pUS202. The iGEM provided backbone was not accetpable as it used an antibiotic resistance gene that did not fit with our validation design. This backbone tested as high copy and therefore did not serve our needs. The second attempt was to move our biobrick by restriction digestion, phorphorylation and blunt end ligation into pSB6A1 which had had its rfp digested out and was de-phosphorylated. This was not succesful and time constraints pushed us to option three which was to use the biobrick in the high copy PSB1C3 that we submitted to the library. <br><br>
 
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The validation design uses three plasmids to form a working integron. There is an integrase plasmid (pUS42), and atti site plasmid (pUS44) and a plasmid with a cassette (i.e. attc sites) (pUS25 and pUS23), all of which were present in the lab we worked in and we proved work together to express the resistance of the cassette which had not been expressed before the combination of these plasmids in one call.  All of these plasmids were in the JM109 strain of E. coli and all validation work was performed in this strain because top10 – the strain we used for making the plasmids has streptomycin resistance in its chromosome and would have given false positives fro the presence of plasmids. <br> <br>
 
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Once these parts were proven to work together our integrase (in whichever form) was to besubstituted for pUS42 which had intigrase expressed constitutivly to prove it worked (i.e. made the genes of the cassette express) and that this expression was controllable. If integrase was proven we hoped to substitute in the casette we made (aaedB and Gm) in for pUS25/23 and have the system opperating with 2 pats we’d made and only one from the lab.
 
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Latest revision as of 18:39, 17 October 2014

iGEM_Link


pUS201 Low Copy Plasmid

Aim

To produce a low copy plasmid backbone for the cloning of integron integrase (IntI1). The integron integrase (IntI1) is believed to be toxic to cells when expressed at high concentrations, thus, it was expected that it would benefit the stability of the integrase construct if it was both controllable, and expressed on a low copy plasmid backbone.

Back to top

Approach

We sought to improve one existing low-moderate copy backbone, pSB6A1, for this purpose. pSB6A1 is derived from pBR332, having a pMB1 ori site, a Rop gene that acts to reduce copy number and ampicillin resistance as the selectable marker. Additionally, pSB6A1 has had a biobrick prefix/suffix cloning site suffix cloning region added, which has disrupted the tetracycline resistance gene present in pBR332.

A large region of defective TetR gene sequence is still remaining, and the cloning region lacks terminators either side of the prefix and suffix. Thus, we sought to assemble a new plasmid, pUS201, incorporating the AmpR, Ori and rop sites from pSB6A1, and the biobrick prefix/suffix cloning region with terminators from pSB1C3.

Figure 1 – General strategy behind the construction of pUS201.

Back to top

Materials

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Method

PCR of the pSB6A1 backbone (from pSB6A1 containing BBa_J04450 was done using primers 1401 and 1402. Similarly, PCR of pSB1C3 (from pSB1C3 containing BBa_K59009 yielded the biobrick cloning region. The overlap between the sequences of the PCR products allowed for Gibson assembly of the final plasmid, pUS201. This was transformed into Top10 E. coli, with the correct clone selected for on ampicillin, with a white colouration.

Figure 2 – Digestion of the plasmid preparation of pUS201 with a variety of restriction endonucleases yields the expected fragment sizes when visualised with agarose gel electrophoresis. The results are as expected providing us with good evidence that the part is what we want.

Back to top

Validation

We aimed to validate that pUS201 was low copy by observing the proliferation of pUS201 positive E. coli in different concentrations of ampicillin or carbenicillin. Unfortunately, the results indicated that pUS201 was not low copy, with the rate of proliferation of bacteria in solutions of LB and carbenicillin similar to pUC19, a high copy plasmid that expresses ampicillin resistance.

Figure 3 – Proliferation of E. coli after 2.5 hours in LB media with various concentrations of carbenicillin, as measured by OD600. Error bars represent the standard error of the mean. pUS201 seems to behave similarly to pUC19, the high copy plasmid, as opposed to the low copy pSB6A1.

Back to top

Retrieved from "http://2014.igem.org/Team:USyd-Australia/pUS201"