From 2007.igem.org

Projects

Design: Wet Lab

Design: Printer

Design: Software

Testing

Construction: The Wetlab

Protocols

Final Result of E.co Lisa

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 plan 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. These are cells that are competent to be transformed in our crazy biological experiments, in case you didn't know. We were having some real trouble with some of our constructions, getting full lawns of cells after a ligation. Now this would be excellent, were it not for the sheer overwhelming odds that something was going seriously wrong. After 3 weeks of testing everything and anything we found that our cells, for some reason, had an innate 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 promoter - R0084 lux pR AHL Promoter - R0062 lac TS Promoter - R0011

lac TS Gene - J06501 tetRPromoter - R0040 Ribosome Binding Site - B0034

RBS.GFP.Terminators - B0015 tetRPromoter - I13504 RNA Lock 1 - J01080

ccdB Gene, Amp/Chlor resistant plasmid - p1010AC ompC/ompF - R0082 ompC/ompF - R0083

Ribosome Binding Site . luxR receiver - #S01414 I13453.rbs.I15008 - S03410

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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

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

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

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

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

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

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

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

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

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

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

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

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

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