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('gBlockDesign');">Design of IntI1 gBlock</h2></li>
 
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The code for the Inti1 gene was sourced from pUS2056 (theUSyd  Coleman- Holmes lab had this LINK). We tried to get the part from the Paris Bettencourt team (from 2010 part <a href="http://parts.igem.org/cgi/partsdb/puttext.cgi">BBa_K329014</a>) but this was not possible so we had to make it. It was modified by Tom Geddes, Rokiah Alford and Nick Coleman to include: <ol>
 
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<li>- A different ribosome binding site, </li>
 
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<li>- To remove an illegal Spe1 site near the end, </li>
 
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<li>- To include a Sac1 restriction enzyme site at the beginning before the IntI1 gene so it could be excised independently of the promoter, </li>
 
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<li>- To remove the pC promoter that was coded in the opposite direction to the promoter we wanted to use and would have clashed with the controllable promotion of this part, and </li>
 
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<li>- To have Gibson ends that overlapped with the backbone we wanted to put it into which is described below.<li>
 
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This backbone, made by honours student Sam Ross, is PSB1C3 with araC-pBAD (which was sourced from <a "href=http://parts.igem.org/Part:BBa_K731201">BBa_K731201</a> a well characterized part) in between the suffix and prefix and will be called SamR’s plasmid throughout our documents. The code for the BioBrick section of this plasmid was used to predict the sequence for junction PCR sequences and our BioBrick’s sequence. </li>
 
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Our first attempt to Gibson Assemble these two – the Inti1 Gblock and SamR’s plasmid – failed because the gibson end that overlapped the araC-pBAD section was incorrect. There was extra code after the code for pBAD which we had not anticipated. This triggered the redesign of the gblock.
 
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<li style="font-weight: bold;"> <h2 onclick="toggle_visibility('gBlockDesign2');">Re-Design of first IntI1 gBlock</h2></li>
 
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Three major changes happened to the gblock. We changed the code for the ribosome cut site which had one base wrong, we decided to put an Nhe1 restriction enzyme cut site between the inti1 gene and the promoters on the SamR construct because the low copy backbone we planned to move this biobrick to for validation had an Sac1 cut site in its backbone, and finally we totally overhauled the Gibson end for the junction between the pBAD promoter and the Inti1 gene because there was extra code on the SamR construct that we didn’t know about when designing this gblock the first time. These changed were performed by our supervisor Nick Coleman in consultation with Rokiah Alford. <br><br>
 
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The gibson assembly of SamR’s plasmid and the Inti1 gblock was successful as was checked in the following ways: junction PCR and sequencing, restriction digestion, biobrick PCR ad sequencing.
 
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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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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>
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<img src="https://static.igem.org/mediawiki/2014/5/5c/USYD2014_pUS201strategy.PNG" width="90%">
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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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The IntI1 gBlock was inserted into the SamR construct using <a href="http://www.addgene.org/plasmid-protocols/gibson-assembly/">Gibson Asssembly</a>. The ends of the gBlock in the redesign of the gBlock were compatible with the ends of the SamR construct and after running the Gibson Assembly reaction we used a variety of methods to prove that our gBlock had inserted correctly (See Below <a href="Proof">Proof</a> section)
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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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<h3>Method</h3>
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<li style="font-weight: bold;"> <h2 onclick="toggle_visibility('Proof');">Proof</h2></li>
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Junction primer PCR:
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The colonies from the Gibson Assembly reaction of the SamR plasmid and the Inti1 gblock that grew of the LB+ Cm25 plates were screened using two sets of junction primers. One set was for the junction between the promoter and the gblock, the second for the junction of the gblock and the PSB1C3 backbone. <br><br>
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Junction primers for the junction of promoter and gblock were iGEM1413 and iGEM1414. These yielded a fragment size of 377bp which was found in 9 out 12 of colonies sampled and patch plated. This result was far more than we anticipated because the selection method was only or the backbone’s resistence not the insert which was only the modified integrase gene. Samples from the patch plate of these positive colonies were subjected to a second junction primer screening. The junction primers for this were iGEM1416 and iGEM25 (last year’s USyd iGEM team designed this primer to amplify the whole biobrick from the suffix end and we re-purposed it here). These yielded a fragment size of 432bp in 8 of the expected 9 colonies that were positive for the 1st junction screen. From the colonies positive for both junction screens 3 were plasmid prep’d (colonies 3, 9, and 33). <br><br>
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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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<h3>Junction primer sequencing:</h3><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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The products of both junction PCRs of these three colonies were sequenced and all sequences (the forward and reverse directions for each of the junctions i.e. 4 sequences per colony) corresponded to that predicted for the junctions using the code of the SamR plasmid and the gblock we designed. <br><br>
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<h3>Plasmid Prep and check with restriction digestion:</h3><br>
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These three plasmid preps were checked by restriction digestion using combinations of an enzyme from the suffix (Spe1 and Pst1) and prefix (EcoR1), as well as Sac1 which has no cut sites and nhe1, an enzyme that cut only once at a site we introduced into the gblock for this purpose and to allow others to move the inti1 gene independently of the rest of the biobrick. The results were as predicted using snap gene for all except the Sac1 digest. We were satisfied with these results despite the Sac1 error because the other fragments were as expected and most importantly because Nhe1 cut, which could only have occurred if the gblock had been incorporated into the plasmid. These results supported the junction primer results.  The enzyme in the Sac1 digest of pUS203 was heat killed and this  digested  plasmid prep was used for the PCR of the biobrick. This showed that the unexpected action of the Sac1 seen in the gel for the digest of this plasmid luckily didn’t reflect an error in the biobrick code. <br><br>
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<h3>Biobrick PCR and sequencing:</h3><br>
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The final check for this plasmid was to PCR the entire biobrick fragment using iGEM16 and iGEM25 (last year’s USyd team’s primers for the biobrick). This yelided a fragment size of 2676bp in 6 out of 8 PCR reactions of heat killed Sac1 restriction digested pUS203.  This PCR product was sequenced using the iGEM16 and iGEM25.
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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.

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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.

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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.

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