Calgary/testing

From 2007.igem.org

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   <li style="margin-bottom:20px;">Third major issue came with one of the pivotal parts of our system, the light sensor. The registry part, M30109, is based off of the light sensor plasmids developped by the Austin iGEM '05 ad '06 teams. This part consists of the three seperate genes, which should form a working light sensor when transformed. Our current theory (as put forth by Patrick King) is that there may be a mutation at one of the biobrick restriction sites that prevented us from being able to isolate the part from its plasmid backbone. The biobrick primers we use also anneal at the biobrick restriction sites, and our attempts to PCR out the part were also unsuccesful. So we spent a few agonizing months trying to figure out why the part would not cut, and would not replicate in PCR, the way it should. Eventually we resorted to contacting the texas team and asking them for the two plasmids from their original project. It was here that we were informed of a potentially significant issue. The texas team had actually co transformed two seperate plasmids into their cells. One texas plasmid contained two of the light sensor genes, while the other contained the third. Apparently, the plasmid with two genes (pPlPCB, with genes Ho1 and PcyA) is unstable, perhaps due to the genes using too much cellular iron from transformed cells and making the cell unhealthy. The registry part had all three of them in one plasmid (M30109), and if pPLPCB is unstable then M30109 should be also. This may have also contributed to our problems.</li>
   <li style="margin-bottom:20px;">Third major issue came with one of the pivotal parts of our system, the light sensor. The registry part, M30109, is based off of the light sensor plasmids developped by the Austin iGEM '05 ad '06 teams. This part consists of the three seperate genes, which should form a working light sensor when transformed. Our current theory (as put forth by Patrick King) is that there may be a mutation at one of the biobrick restriction sites that prevented us from being able to isolate the part from its plasmid backbone. The biobrick primers we use also anneal at the biobrick restriction sites, and our attempts to PCR out the part were also unsuccesful. So we spent a few agonizing months trying to figure out why the part would not cut, and would not replicate in PCR, the way it should. Eventually we resorted to contacting the texas team and asking them for the two plasmids from their original project. It was here that we were informed of a potentially significant issue. The texas team had actually co transformed two seperate plasmids into their cells. One texas plasmid contained two of the light sensor genes, while the other contained the third. Apparently, the plasmid with two genes (pPlPCB, with genes Ho1 and PcyA) is unstable, perhaps due to the genes using too much cellular iron from transformed cells and making the cell unhealthy. The registry part had all three of them in one plasmid (M30109), and if pPLPCB is unstable then M30109 should be also. This may have also contributed to our problems.</li>
</ul>
</ul>
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<p><a style="text-decoration:none" name="top"> So with the introduction and important stuff out of the way check out our part by part testing section. Also click on any part name to link to our simplified table of parts! </a></p>
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<p><a style="text-decoration:none; color:#000000" name="top"> So with the introduction and important stuff out of the way check out our part by part testing section. Also click on any part name to link to our simplified table of parts! </a></p>
<table width="100%" style="margin-bottom:30px;">
<table width="100%" style="margin-bottom:30px;">
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     <td valign="top"><ul id="sub">
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         <li><a href="#R0084" title="ompF promoter R0084"> ompF promoter R0084 </a></li>
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         <li><a href="#R0084" title="ompF promoter R0084"> <em>ompF</em> promoter - R0084 </a></li>
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         <li><a href="#R0062" title="AHL Promoter R0062"> AHL Promoter R0062</a></li>
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         <li><a href="#R0062" title="AHL Promoter R0062"> <em>lux pR </em>AHL Promoter - R0062</a></li>
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         <li><a href="#R0011" title="Lac TS Promoter R0011"> Lac TS Promoter R0011 </a></li>
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         <li><a href="#R0011" title="Lac TS Promoter R0011"> <em>lac</em> TS Promoter - R0011 </a></li>
       </ul></td>
       </ul></td>
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     <td valign="top"><ul id="sub">

Revision as of 22:07, 23 October 2007

The Primers have been designed the parts have been ordered and a plan has been put together. Now its time to start construction right? Wrong! All of these parts and ideas have to be thoroughly tested. We need to make sure that all the parts we've ordered are what they're supposed to be, where they're supposed to be and do what they are supposed to do. Tedious I know...

This portion of our project provided unique challenges for a couple of reasons, which we are going to outline here so that anyone attempting to repeat our project won't have to deal with the same intense frustration and agony we did. Aren't we nice?

Some of our bigger challenges in the testing phase...

  • Ok, first of all. When we ordered our initial batch of parts from MIT we tried to start working with them right away. This was not a great idea as full descriptions of each parts coordinates on the plates were not readily available from the registry. This lead to a brilliantly doomed attempt at reverse engineering the part locations. Things never really worked out with this batch unfortunately, did I mention it was brilliantly doomed. So The lesson is to be very careful with the parts you have ordered and take the time to work with the registry and ensure that you know 100% where everything is and how it is labled. Learn from us... seriously
  • Our second major hurdle had to do with our TOP10 cells. Those are cells that are competent to be transformed in our crazy biological experiments in case you didn't know. Our cells, for some reason had an inate resistance to chloramphenicol. This was a huge issue as most of our test plates contained chloramphenicol which we were using as a selection pressure. So while the odds of this happening are unlikely keep in mind this could be one of the problems you may face.
  • Third major issue came with one of the pivotal parts of our system, the light sensor. The registry part, M30109, is based off of the light sensor plasmids developped by the Austin iGEM '05 ad '06 teams. This part consists of the three seperate genes, which should form a working light sensor when transformed. Our current theory (as put forth by Patrick King) is that there may be a mutation at one of the biobrick restriction sites that prevented us from being able to isolate the part from its plasmid backbone. The biobrick primers we use also anneal at the biobrick restriction sites, and our attempts to PCR out the part were also unsuccesful. So we spent a few agonizing months trying to figure out why the part would not cut, and would not replicate in PCR, the way it should. Eventually we resorted to contacting the texas team and asking them for the two plasmids from their original project. It was here that we were informed of a potentially significant issue. The texas team had actually co transformed two seperate plasmids into their cells. One texas plasmid contained two of the light sensor genes, while the other contained the third. Apparently, the plasmid with two genes (pPlPCB, with genes Ho1 and PcyA) is unstable, perhaps due to the genes using too much cellular iron from transformed cells and making the cell unhealthy. The registry part had all three of them in one plasmid (M30109), and if pPLPCB is unstable then M30109 should be also. This may have also contributed to our problems.

So with the introduction and important stuff out of the way check out our part by part testing section. Also click on any part name to link to our simplified table of parts!


ompF promotor

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Part R0084
Plasmid pSB1A2
Description ompF promoter
Source Plate
Transformed YES
PCR Verified June 29th 2007
Glycerol YES
Length (base pairs) 108
Labled Gel from June 29th 2007

lux pR AHL promotor

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Part R0062
Plasmid pSB1A2
Description lux pR AHL promotor
Source Glycerol
Transformed YES
PCR Verified June 29th 2007
Glycerol YES
Length (base pairs) 55
Labled Gel from June 29th 2007

lac TS promoter

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Part R0011
Plasmid pSB1A2
Description lac TS promoter
Source Plate
Transformed YES
PCR Verified June 29th 2007
Glycerol YES
Length (base pairs) 55
Labled Gel from June 29th 2007

lac TS Gene

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Part J06501
Plasmid pSB1A2
Description lac TS Gene
Source Plate
Transformed YES
PCR Verified July 5th 2007
Glycerol YES
Length (base pairs) 1153
Labled Gel from July 5th 2007

tetRPromoter

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Part R0040
Plasmid pSB1A2
Description tetR promoter (constitutive)
Source Plate
Transformed YES
PCR Verified July 9th 2007
Glycerol YES
Length (base pairs) 54
Labled Gel from July 9th 2007

Ribosome Binding Site

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Part B0034
Plasmid c
Description Ribosome binding site
Source Glycerol
Transformed YES
PCR Verified July 9th 2007
Glycerol YES
Length (base pairs) 12
Labled Gel from July 9th 2007

Double Terminator

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Part B0015
Plasmid pSB1AK3
Description Double terminator
Source Glycerol
Transformed YES
PCR Verified July 9th 2007
Glycerol YES
Length (base pairs) 129
Labled Gel from July 9th 2007

RBS.GFP.Terminators

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Part I12504
Plasmid pSB1A2
Description RBS.GFP.Terminators
Source Glycerol
Transformed YES
PCR Verified July 9th 2007
Glycerol YES
Length (base pairs) 875
Labled Gel from July 9th 2007

RNA Lock 1

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Part J01080
Plasmid n.a
Description RNA Lock. Normally locked, bloacks acces to RBS
Source Synthesis
Transformed YES
PCR Verified July 26th 2007
Glycerol YES
Length (base pairs) 40
Labled Gel from July 26th 2007

ccdB Gene, Amp/Chlor resistant plasmid

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Part p1010 AC
Plasmid pSB1AC3
Description ccdB Gene, Amp/Chlor resistant plasmid
Source Plate
Transformed YES
PCR Verified July 26th 2007
Glycerol YES
Length (base pairs) 675
Labled Gel from July 26th 2007

ompC/ompF

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Part R0082
Plasmid pSB1A2
Description ompC/ompF
Source Plate
Transformed YES
PCR Verified July 26th 2007
Glycerol YES
Length (base pairs) 108
Labled Gel from July 26th 2007

ompC/ompF - R0083

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Part R0083
Plasmid pSB1A2
Description ompC/ompF
Source Plate
Transformed YES
PCR Verified July 26th 2007
Glycerol YES
Length (base pairs) 78
Labled Gel from July 26th 2007

Ribosome Binding Site . luxR receiver

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Part S01414
Plasmid pSB1A2
Description Ribosome Binding Site . luxR receiver
Source Plate
Transformed YES
PCR Verified July 26th 2007
Glycerol YES
Length (base pairs) 799
Labled Gel from July 26th 2007

I13453.rbs.I15008

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Part S03410
Plasmid pSB1AT3
Description I13453.rbs.I15008
Source Plate
Transformed NO
PCR Verified July 31st 2007
Glycerol NO
Length (base pairs) 907
Labled Gel from July 26th 2007