Team:Imperial College London/Human/Containment

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<h1>Human Practices</h1>
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<h1>GM Release</h1>
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<h1>Containment</h1>
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<p>There are many different biological aspects that need to be taken into account when deliberating release of genetically modified organisms into the environment. A big issue associated with this is containment of genetic information and preventing its spread to other organisms. To prevent spread of our auxin-producing plasmid in the environment, we have devised Gene Guard, a containment device that will actively prevent horizontal gene transfer. In addition, we have chosen <i>Escherichia coli</i> as our chassis to prevent spread to adjacent ecosystems.</p>
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<p>To prevent spread of the auxin-producing plasmid in the environment, we have devised a containment device that will be able to kill other bacteria that take up the plasmid. In addition, we have devised experiments to test the survivability of E. coli in soil to evaluate whether these bacteria would be outcompeted by other soil microorganisms. Soil-dwelling protozoa also play an important role as they have been shown to feed off bacteria.</p>
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<h2>Soil Experiment</h2>
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<p>This experiment is designed to determine the survivability of E. coli in soil. If bacteria were to be left in the soil we can estimate accurately the length of time they will be alive by carrying out this experiment. It is probable that the bacteria will live for longer in sterile soil than non-sterile soil, due to factors such as competition or they are being attacked by soil bacteria. This experiment was started by our work experience student Kiran and carried on by us after he left. </p>
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<p>For this experiment, we placed soaked small discs of filter paper in GFP expressing E. coli and put these into autoclaved and non-autoclaved soil. The bacteria were incubated for 10 days. Cultures were grown up in ampicillin and/or kanamycin containing medium every day to measure the optical density and thus determine how many bacteria had survived in sterile and non-sterile soil for set periods of time.</p>
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<p>To ensure that we were not growing up antibiotic-resistant soil bacteria, we grew up cultures from non-inoculated soil as negative controls. In addition, we grew up cultures on ampicillin and kanamycin containing plates on day 6 to check for fluorescence. There were no colonies detected on the negative control plates, showing that OD measurements in the negative control were due to soil particles in the media. The colonies grown up from E coli inoculated in soil were brightly fluorescent, confirming the presence of plasmid-carrying E coli in the soil after a long period of time.</p>
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<p>Subsequently, we stopped measuring the OD of bacteria and started plating out cultures from sterile and non-sterile media to check for presence of the plasmid.</p>
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<p>
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<h3>Results</h3>
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<p>Non-Sterile:<br><br>
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1A 0.315<br>
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1B 0.393<br>
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1C 0.361<br>
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2A 0.553<br>
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2B 0.583<br>
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2C 0.548<br>
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3A 0.399<br>
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3B 0.668<br>
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3C 0.300<br><br>
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Sterile<br><br>
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1A O.523<br>
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1B 0.548<br>
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1C 0.476<br>
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2A 0.716<br>
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2B 0.616<br>
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2C 0.664<br>
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3A 0.950<br>
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3B 0.887<br>
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3C 1.002</p>
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<h2>Containment Device</h2>
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<h2>Gene Guard design</h2>
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<p><u>1. Goal</u> Bacteria frequently pass on genetic information through horizontal gene transfer. This can happen in a variety of ways, including transfer or a mobile plasmid into another bacterium (1). As a result, a significant portion of bacterial genomes (12.8% of <i>Escherichia coli</i> genomes) consists of foreign DNA (2). If the DNA we engineered into our bacteria were to be transferred to plant pathogenic bacteria, it may give them a competitive advantage. This could have detrimental consequences and negatively affect plants and the ecosystem. Therefore, we want to prevent spreading of the genetic information we have engineered into our chassis. </p>
 +
<p><u>2. Action</u> Early on in the project, we recognised that containment of genetic information is an issue that has to be taken very seriously. We discussed many different ways in which we could ensure that the DNA we engineer into our bacteria does not spread to other microorganisms or adjacent ecosystems. We consulted our advisors in the synthetic biology centre at Imperial who informed us about already existing "kill switches". Most of these are based on killing bacteria in response to a specific stimulus. However, many of the compounds that cause activation of these switches could not be applied to soil without side effects. In addition, we could never ensure that all bacteria in the soil would be eradicated through application of these compounds. Switches that kill bacteria after a certain number of replication cycles are also not necessarily applicable as the lysed bacteria leave behind genetic information that can be taken up by other soil microorganisms. </p>
 +
<p><u>3. Result</u> We took a novel approach towards designing a "kill switch" and designed a containment device that is able to contain genetic information in our bacteria at the same time as preventing horizontal gene transfer to other microorganisms. <b>Gene Guard</b> is based on a toxin/anti-toxin system taken from the lysis cassette made by the Berkeley 2008 team. This originally involved the secretion of holin along with lysozyme, so that the holin would form pores in the inner membrane and allow the lysozyme to break down the cell wall. This caused the cell to lyse when the inducible promoter was induced by arabinose.
 +
Also included in this cassette is the gene for antiholin, under the control of a weak, constitutive promoter to prevent any leakage. Our project takes this a step further. By using the anti-holin's ability to inhibit the activity of holin, we can create a system in which the lytic activity of holin can be negated by the presence of antiholin in certain cells. This means that we can make a plasmid that can kill one cell, but replicate in another.</p>
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<p>The containment device is based on a toxin/anti-toxin system taken from the lysis cassette made by the Berkeley 2008 team. This originally involved the secretion of holin along with lysozyme, so that the holin would form pores in the inner membrane and allow the lysozyme to break down the cell wall. This will cause the cell to lyse when the inducible promoter was induced by arabinose.</p>
 
-
<p>
 
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Also included in this cassette is the gene for antiholin, under the control of a weak, constitutive promoter to prevent any leakage.</p>
 
-
<p>
 
-
Our project takes this a step further. By using the anti-holin's ability to inhibit the activity of holin, we can create a system in which the lytic activity of holin can be negated by the presence of antiholin in certain cells. This means that we can make a plasmid that can kill one cell, but replicate in another.</p>
 
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<h2>Panel</h2>
 
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<p>As  part of our human practices process, we held two panels. The first panel consisted of Prof Richard Kitney, Dr Tom Ellis, Dr Guy-Bart Stan, Charlotte Jarvis and Kirsten Jensen.</p>
 
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<h1>Chassis choice</h1>
 +
<p><u>1. Goal</u> In chassis choice, we had to consider several aspects. We wanted to choose a chassis that we would be able to transport to arid areas, preferably already enveloped inside a solid seed coat. In addition, we want the bacteria to be able to persist in the soil long enough to carry out their function. On the other hand, we also want to prevent spread of the bacteria into far-away ecosystems where they are more likely to have a detrimental effect on the ecological balance. </p>
 +
<p><u>2. Action</u>
 +
We consulted two ecologists, who are experts in above-below ground interactions and soil microbial ecology. They both advised us that while it may be more obvious to use naturally occurring soil bacteria such as <i>Bacillus subtilis</i>, <i>Escherichia coli</i> is less likely to survive in soil and may ensure better containment. <b>Dr Alexandru Milcu</b> pointed out that this is especially important considering that very high auxin secretion may skew plant populations. While this is not an issue in areas where the ecosystem is already badly affected, spread to other ecosystems, especially via spores, is a big issue. <b>Dr Robert Griffiths</b> also advised us that while engineering naturally occurring soil bacteria might lead to better persistence and cause our project to be more efficient, containment would be more easily achieved by using bacteria that do not normally occur in soil such as <i>E. coli</i> as they are more likely to be outcompeted.</p>
 +
<p>These arguments caused us to pin-point our chassis choice on <i>B. subtilis</i>, a natural spore-forming bacterium that naturally occurs in soil and <i>E. coli</i>. We initially codon-optimised our genes for both of these species. At the first human practices panel, we thoroughly discussed the advantages and disadvantages associated with both chassis choices (Figure 1).</p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/3/34/ICL_2011_chassis_choice.png" width=400/></p>
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<p><i>Figure 1. Advantages and disadvantages of possible AuxIn chassis</i></p>
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<p><u>3. Result</u> Containment and possible contamination of other areas is a very big human practices issue. With <i>B. subtilis</i> as our chassis we would never be able to ensure complete containment. On the other hand, enveloping <i>E. coli</i> in a seed coat is mostly  a mechanical issue that we should be able to overcome. We therefore chose to use <i>E coli</i> as the chassis for AuxIn.</p>
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<p>The panel addressed many different questions that we later used to inform our design.</p>
 
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<p><b>Could the bacteria impact the germination of the seeds?</b><br>
 
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The coat itself would not be prohibiting germination. It is possible to design the coat sufficiently well to ensure that this would not happen. In addition, seeds normally germinate in soil full of bacteria that do not prevent germination.</p>
 
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<p><b>How can we ensure that the auxin does not kill the plants?</b><br>
 
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We will be able to vary the inoculum of bacteria in the coat. We will get an experimental estimate of the auxin production, which will help us estimate the ideal number of bacteria to be contained in the seed coat. While a weak promoter may be better for constitutive expression of auxin, it will be easier to weaken the promoter later. We have used an insulator sequence to separate the promoter from the RBS so that we will  be able to replace the promoter very easily. This may also contribute to the fine-tuning of expression and thus help us make sure that auxin is expressed at ideal concentrations. The worst case scenario consists of the auxin producing genes being transferred to other bacteria that become pathogenic. However, this could be tested exclusively beforehand and the infrastructure for this separate development and safety testing stage is already in place. In addition, unlike synthetic auxin, natural auxins such as IAA have a short half-life and degrade rapidly. </p>
 
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<p><b>What is the risk-benefit relationship of our implementation?</b><br>
 
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In our implementation, we are trying to improve already existing practices. We do have to take a certain risk to combat desertification. However, is putting GM bacteria into soil worth speeding up the acacia tree planting process? How much does this really help? While GM bacteria may pose a risk, introducing foreign plant species that also show drought resistance and grow faster than acacia trees can be extremely risky and introduction of foreign species into ecosystems has already had negative consequences all over the world. This effect is likely to be worsened by the fact that we would be introducing the foreign species into an already damaged ecosystem. We may also be able to plant other fast growing plants at the same time as planting our coated seeds to hold the soil down while the seeds are growing.</p>
 
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<p><b>Should we be using <i>B. subtilis</i> or <i>E. coli</i> as our chassis?</b><br>
 
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<i>B. subtilis</i> spores spread very easily over long distances and may thus be blown into different ecosystems where they may have negative effects. On the other hand, its spores would be easier to integrate into a seed coat. <i>E. coli</i> is not as easy to integrate into the seed coat. However, it does not form spores and is therefore very likely to stay inside the ecosystem we introduce the microbes into. We have already shown that <i>E. coli</i> is able to survive in non-sterile soil for more than two weeks and that it can pass on its plasmid to other bacteria, enabling them to express GFP and antibiotic resistance. By using <i>E. coli</i> as our chassis, we can be sure that the bacteria will survive in the soil for a reasonably long period of time but not spread as rapidly as <i>B. subtilis</i> spores would. At the same time, we will be preventing plasmid transfer using the BacTrap. We should be able to overcome the technical challenge of putting <i>E. coli</i> into the seed coat.
 
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<p><b>Would we be able to get rid of the bacteria once they are in the soil?</b><br>
 
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The kill switch is never 100% effective and the bacteria will lose the plasmid. In addition, bacteria killed by kill switches still leave behind DNA that can be conjugated by other, naturally occurring bacteria. We may not be able to take the bacteria back out of the environment after they have been distributed into soil. Instead, we will be aiming to prevent spread of the plasmid. We will be using <i>E. coli</i> as our chassis, which should be outcompeted in the soil and our BacTrap device will be used to prevent plasmid conjugation.
 
 +
<h2>Containment vs. bacterial survival</h2>
 +
<p><u>1. Goal</u> One of the main problems associated with using an <i>E. coli</i> (lab) strain as our chassis is its presumed inability to survive in soil for a long time due to harsh competition in the rhizosphere. Although <i>E. coli</i> have been shown to be able to survive in soil for significant amounts of time (more than 130 days in the case of the pathogenic strain O175, as shown by Maule, 2000 (3)), the ability to survive even in manure sludge varies greatly between different strains (4). We want our bacteria to survive in the soil and retain their plasmids for long enough to carry out their function but we also don't want the GM modified bacteria to become established.
 +
<p><u>2. Action</u> To investigate the survival of lab strains of <i>E. coli</i> in soil, we set up a separate experiment. This experiment was started by our work experience student Kiran and carried on by us after he left. </p>
 +
<p>We placed soaked small discs of filter paper in GFP expressing E. coli and put these into autoclaved and non-autoclaved soil. The bacteria were incubated for over six weeks. Cultures were grown up in medium containing both ampicillin and kanamycin every week. These cultures were plated out and checked for fluorescence to check for presence of the plasmid. To ensure that we were not growing up antibiotic-resistant soil bacteria, we grew up cultures from non-inoculated soil as negative controls.</p>
 +
<p><u>3. Result</u> After three weeks, the bacteria were still growing in non-sterile media and expressing GFP. In addition, there were bacteria present on the plates that were resistant to ampicillin and kanamycin and some of them expressed GFP. It is therefore very likely that the GFP-expressing bacteria passed on the GFP-expressing plasmid to other bacteria in their environment. After six weeks, fluorescence could no longer be detected on the plates grown up from non-sterile soil cultures.</p>
 +
<p>References:<br>
 +
(1) Ochman, H. et al. (2000) Lateral gene transfer and the nature of bacterial innovation. <i>Nature</i> 405, pp. 299-305.<br>
 +
(2) Thomas, C. & Nielsen, K. (2005) Mechanisms of, and Barriers to, Horizontal Gene Transfer between Bacteria. <i>Nature Reviews Microbiology</i> 3, pp. 711-721.<br>
 +
(3) Maule, A. (2000) Survival of verocytotoxigenic <i>E. coli</i> O175 in soil, water and on surfaces. <i>Symp Ser Soc Appl Microbiol </i><b>29:71-78.</b><br>
 +
(4) Topp, E. et al. (2006) Strain-dependent variability in growth and survival of <i>Escherichia coli</i> in agricultural soil. <i>FEMS Microbiology Ecology</i><b> 44:303-308.</b></p>
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Latest revision as of 11:10, 15 September 2011




Informing Design

We consulted numerous experts in various fields to ensure that the design of the AuxIn system respects all relevant social, ethical and legal issues. One module of our system, Gene Guard, is a direct result of brainstorming around the issues involved in the release of genetically modified organisms (GMOs). Although we have only reached the proof of concept stage, we have put a lot of thought into how AuxIn may be implemented as a product and the legal issues that would be involved.




GM Release

There are many different biological aspects that need to be taken into account when deliberating release of genetically modified organisms into the environment. A big issue associated with this is containment of genetic information and preventing its spread to other organisms. To prevent spread of our auxin-producing plasmid in the environment, we have devised Gene Guard, a containment device that will actively prevent horizontal gene transfer. In addition, we have chosen Escherichia coli as our chassis to prevent spread to adjacent ecosystems.

Gene Guard design

1. Goal Bacteria frequently pass on genetic information through horizontal gene transfer. This can happen in a variety of ways, including transfer or a mobile plasmid into another bacterium (1). As a result, a significant portion of bacterial genomes (12.8% of Escherichia coli genomes) consists of foreign DNA (2). If the DNA we engineered into our bacteria were to be transferred to plant pathogenic bacteria, it may give them a competitive advantage. This could have detrimental consequences and negatively affect plants and the ecosystem. Therefore, we want to prevent spreading of the genetic information we have engineered into our chassis.

2. Action Early on in the project, we recognised that containment of genetic information is an issue that has to be taken very seriously. We discussed many different ways in which we could ensure that the DNA we engineer into our bacteria does not spread to other microorganisms or adjacent ecosystems. We consulted our advisors in the synthetic biology centre at Imperial who informed us about already existing "kill switches". Most of these are based on killing bacteria in response to a specific stimulus. However, many of the compounds that cause activation of these switches could not be applied to soil without side effects. In addition, we could never ensure that all bacteria in the soil would be eradicated through application of these compounds. Switches that kill bacteria after a certain number of replication cycles are also not necessarily applicable as the lysed bacteria leave behind genetic information that can be taken up by other soil microorganisms.

3. Result We took a novel approach towards designing a "kill switch" and designed a containment device that is able to contain genetic information in our bacteria at the same time as preventing horizontal gene transfer to other microorganisms. Gene Guard is based on a toxin/anti-toxin system taken from the lysis cassette made by the Berkeley 2008 team. This originally involved the secretion of holin along with lysozyme, so that the holin would form pores in the inner membrane and allow the lysozyme to break down the cell wall. This caused the cell to lyse when the inducible promoter was induced by arabinose. Also included in this cassette is the gene for antiholin, under the control of a weak, constitutive promoter to prevent any leakage. Our project takes this a step further. By using the anti-holin's ability to inhibit the activity of holin, we can create a system in which the lytic activity of holin can be negated by the presence of antiholin in certain cells. This means that we can make a plasmid that can kill one cell, but replicate in another.

Chassis choice

1. Goal In chassis choice, we had to consider several aspects. We wanted to choose a chassis that we would be able to transport to arid areas, preferably already enveloped inside a solid seed coat. In addition, we want the bacteria to be able to persist in the soil long enough to carry out their function. On the other hand, we also want to prevent spread of the bacteria into far-away ecosystems where they are more likely to have a detrimental effect on the ecological balance.

2. Action We consulted two ecologists, who are experts in above-below ground interactions and soil microbial ecology. They both advised us that while it may be more obvious to use naturally occurring soil bacteria such as Bacillus subtilis, Escherichia coli is less likely to survive in soil and may ensure better containment. Dr Alexandru Milcu pointed out that this is especially important considering that very high auxin secretion may skew plant populations. While this is not an issue in areas where the ecosystem is already badly affected, spread to other ecosystems, especially via spores, is a big issue. Dr Robert Griffiths also advised us that while engineering naturally occurring soil bacteria might lead to better persistence and cause our project to be more efficient, containment would be more easily achieved by using bacteria that do not normally occur in soil such as E. coli as they are more likely to be outcompeted.

These arguments caused us to pin-point our chassis choice on B. subtilis, a natural spore-forming bacterium that naturally occurs in soil and E. coli. We initially codon-optimised our genes for both of these species. At the first human practices panel, we thoroughly discussed the advantages and disadvantages associated with both chassis choices (Figure 1).

Figure 1. Advantages and disadvantages of possible AuxIn chassis

3. Result Containment and possible contamination of other areas is a very big human practices issue. With B. subtilis as our chassis we would never be able to ensure complete containment. On the other hand, enveloping E. coli in a seed coat is mostly a mechanical issue that we should be able to overcome. We therefore chose to use E coli as the chassis for AuxIn.

Containment vs. bacterial survival

1. Goal One of the main problems associated with using an E. coli (lab) strain as our chassis is its presumed inability to survive in soil for a long time due to harsh competition in the rhizosphere. Although E. coli have been shown to be able to survive in soil for significant amounts of time (more than 130 days in the case of the pathogenic strain O175, as shown by Maule, 2000 (3)), the ability to survive even in manure sludge varies greatly between different strains (4). We want our bacteria to survive in the soil and retain their plasmids for long enough to carry out their function but we also don't want the GM modified bacteria to become established.

2. Action To investigate the survival of lab strains of E. coli in soil, we set up a separate experiment. This experiment was started by our work experience student Kiran and carried on by us after he left.

We placed soaked small discs of filter paper in GFP expressing E. coli and put these into autoclaved and non-autoclaved soil. The bacteria were incubated for over six weeks. Cultures were grown up in medium containing both ampicillin and kanamycin every week. These cultures were plated out and checked for fluorescence to check for presence of the plasmid. To ensure that we were not growing up antibiotic-resistant soil bacteria, we grew up cultures from non-inoculated soil as negative controls.

3. Result After three weeks, the bacteria were still growing in non-sterile media and expressing GFP. In addition, there were bacteria present on the plates that were resistant to ampicillin and kanamycin and some of them expressed GFP. It is therefore very likely that the GFP-expressing bacteria passed on the GFP-expressing plasmid to other bacteria in their environment. After six weeks, fluorescence could no longer be detected on the plates grown up from non-sterile soil cultures.

References:
(1) Ochman, H. et al. (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405, pp. 299-305.
(2) Thomas, C. & Nielsen, K. (2005) Mechanisms of, and Barriers to, Horizontal Gene Transfer between Bacteria. Nature Reviews Microbiology 3, pp. 711-721.
(3) Maule, A. (2000) Survival of verocytotoxigenic E. coli O175 in soil, water and on surfaces. Symp Ser Soc Appl Microbiol 29:71-78.
(4) Topp, E. et al. (2006) Strain-dependent variability in growth and survival of Escherichia coli in agricultural soil. FEMS Microbiology Ecology 44:303-308.