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Team:Gaston Day School/Project
From 2011.igem.org
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Overall project
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Gaston Day School’s iGEM project for 2011 has two distinct but complementary parts. First, we plan to build a functional nitrate detector using RFP. Red Fluorescent Protein has one distinct advantage over the more traditional Green Fluorescent Protein; it is visible without any special equipment. Our goal is to have a detector that is easy for anyone to use in the field. Most people, including farmers and ranchers, who would need to detect nitrogen pollution will not have a pocket UV light! We envision a kit that could be used to determine if the runoff of a particular farm was high in nitrogen. The kit will include all necessary components for running the test and then decontaminating the resulting growth to prevent release of the engineered bacteria into the environment. The second part of our project involves a close look at the actual risks of accidental release of the engineered bacteria into the environment. Many groups, including ours, have proposed and built environmental detectors of various sorts. Often, these detectors come with sophisticated mechanisms for preventing the release or for preventing the bacteria from growing if released. We would like to include a very simple mechanism for killing or denaturing the bacteria in our detector kit – bleach. Bleach is highly effective at killing bacteria and is readily available to the average person. Even if we include the bleach in the kit, we realize that many people do not (or will not) read and follow directions. We plan to simulate a variety of conditions under which our detector could be introduced into the environment, ranging from simply dumping it in the sink to pouring it into the local creek or soil. By producing survivorship curves, we can estimate the real risk of spreading the recombinant bacteria into the environment. |
Project Details
Part 1: Construction
We built the RFP Nitrate Detector using NEB's BioBrick Construction Kit. We digested the pYeaR promoter (BBa_216005) with EcoRI and SpeI and digested the RFP plasmid (BBa_K081014) with XbaI and PstI. Both initial plasmids were Ampicillin resistant. These digested fragments were mixed and ligated to the provided, liniarized pSB3C1 plasmid. The ligation mix was grown under Chloramphenicol selection. The resulting colonies were tested for nitrate responsive RFP production. A schematic of the process is shown below.
Experiments and Results
During the formation of our nitrate detector, it was necessary to run multiple gels to see if we had good DNA for the ligation. When narrowing down which strain of detector to use we came to the conclusion to use NRFP-2 our second strain of the detector. After testing it with varying millimolar concentrations of calcium nitrate, we saw that it did indeed work and that we were able to pick up its presence at about 10 mM.
As we moved on to the safety part of our project, we thought of multiple ways to find out how safe our nitrate detector really was. We found that after about 5 minutes of 10% bleach, all of the bacteria had died and there was no way to make viable cultures. One way to test its survival in tap water was to take dillutions of about 5mL of our bacteria to 1 liter of tap water and then plate bacteria at 0, 15, 30, 45, and 60 minutes. Our results showed that the plates were uncountable. Even at 1:1000 dillutions of our dillution, the only countable samples were the 30 minute ones. A repeat of the experiment was done of smaller dillutions and showed close to the same results. All dillutions of 1:1000 were now countable. This shows a significante safety issue which can be resolved in the future after more testing.
Part 2: Safety
To ensure that our detector can be sent out to everyone, we needed to make sure that it would do little to harm the environment if not disposed or used properly. Our bacteria carries the antibiotic resistance gene for chloramphenocol and if this was spread somehow, it could result in other kinds of bacteria gaining the same immunity. These bacteria could potentially be dangerous to the environment and possibly humans. One way to know if the bacteria could conjugate would be to assume that someone did not follow the directions and did not kill the bacteria with bleach provided in the kit. This person might have just poured our bacteria down the drain and into the municipal water supply or even just poured it on the ground.