Difference between revisions of "Team:UMaryland/Results"

Revision as of 00:46, 19 September 2015

Plating Tests

Negative Control (No Pressure)

Figure 1. Negative control was K1783002 (constitutive unstable RFP) grown in media without chloramphenicol. After 2-3 days, plates demonstrate fewer than 10 colonies per plate, suggesting that antibiotic resistance is now only minimally present in the culture.

Chloramphenicol Pressure Only

Figure 2. K1783002 (constitutive unstable RFP) grown in media with chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.

Chlor + Hok-Sok Pressure

Figure 3. K1783003 (constitutive unstable RFP under Hok-Sok regulation) grown in media with chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.

Hok-Sok Pressure Only

Figure 4. K1783003 (constitutive unstable RFP under Hok-Sok regulation) grown in media without chloramphenicol. Colonies persist through several days of plating, suggesting that antibiotic resistance was maintained throughout the test.

Growth Curve

Figure 5. Growth curves of parallel cultures grown with and without chloramphenicol. The presence of Hok-Sok on the inserted plasmid does not appear to have a major effect on the bacterial growth rate. This is in line with our qualitative observations, where we do not observe any major difference in cell density during plating or fluorescence studies.

Analysis of Fluorescence Loss

Over time, colonies appear to stop producing RFP.

This effect occurs even when the culture is under chloramphenicol pressure.

This effect occurs much more slowly when the Hok-Sok cassette is adjacent to the RFP construct.

With or without added chloramphenicol, Hok-Sok appears to be able to maintain a relatively constant level of fluorescence.

Figure . Representative gram stain of culture. Gram staining of our samples confirms that our samples are gram-negative bacilli, making it highly unlikely that our cultures have been contaminated.

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 <img src ="https://static.igem.org/mediawiki/2015/1/17/Pinky.png" height="420px" width="420px">
-<p style = "font-size:18px">Red cells are red
+<p style = "font-size:18px">Figure . Representative gram stain of culture. Gram staining of our samples confirms that our samples are gram-negative bacilli, making it highly unlikely that our cultures have been contaminated.</p>
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 <p style = "font-size:24px">Moving Forward</p>
-<p style = "font-size:18px">In the future there is great potential to use the Hok-Sok system both in vitro and in vivo. In the laboratory, using plasmids which contain the Hok-Sok cassette along with some sort of positive selection agent, such as a fluorescent protein, instead of an antibiotic resistance gene would permit selection of colonies containing the desired plasmid without ever using antibiotics. Further the Hok-Sok system could have a transformative role outside the lab. Increasingly synthetic biology projects are creating genetically modified bacteria which are designed for release into the soil, water sources, and ingestion by animals. If these bacteria carried antibiotic resistance genes, there would be ample concern about other bacteria acquiring this resistance through horizontal gene transfer. However, if the Hok-Sok system was used instead as a selecting agent and to maintain plasmids, it would reduce concern over increasing the number of antibiotic resistant bacterial strains.</p>
+<p style = "font-size:18px">In the future there is great potential to use the Hok-Sok system both <i>in vitro</i> and <i>in vivo</i>. In the laboratory, using plasmids which contain the Hok-Sok cassette along with some sort of positive selection marker, such as a fluorescent protein, instead of an antibiotic resistance gene would permit for positive selection of desired colonies without the use of antibiotics.</p>
+<p style = "font-size:18px">Furthermore, the Hok-Sok system could have a transformative role outside the lab. Synthetic biology projects often rely upon genetically modified bacteria which are designed for release into the soil, water sources, and ingestion by animals. Our 2014 project, for example, involved <i>E. coli</i> that could one day detect an oyster pathogen, and <a href = "https://2014.igem.org/Team:Toulouse"> the University of Toulouse 2014 iGEM team</a> was interested in designing bacteria capable of defending trees against fungal attack. If these bacteria carried antibiotic resistance genes, there would be ample concern about other bacteria acquiring this resistance through horizontal gene transfer. However, if the Hok-Sok system was used to maintain plasmids, it would reduce concern over increasing the number of antibiotic resistant bacterial strains.</p>
-<p style = "font-size:18px">Additionally there is potential to take advantage of the Hok-Sok cassette as a method of post-transcriptional control over a variety of genes. As Hok is indirectly regulated by the binding of Sok to Mok directly upsteam of Hok, there is potential to control protein expression through using the interplay of Sok and Mok to prevent the translation of various mRNA transcripts. Although we did not focus on this, there is great potential for experimentation.</p>
+<p style = "font-size:18px">In addition, there is potential to take advantage of the Hok-Sok cassette as a method of post-transcriptional control over a variety of genes. As hok is indirectly regulated by the upstream binding of sok to mok, there is potential to control protein expression through using the interplay of sok and mok to prevent the translation of various mRNA transcripts. Although we did not focus on this, there is great potential for experimentation.</p>
-<p style = "font-size:18px">As mentioned earlier many synthetic biology projects, including those within iGEM intend to release their final product into nature. As our testing supports the conclusion that Hok-Sok can successfully be implemented to maintain plasmids without antibiotic, it would be wonderful and intriguing to test this further ourselves or see other iGEM teams test this by implementing the Hok-Sok cassette as the maintenance system on a project where the final product is intended for release into  nature or animal ingestion. Successful implementation of this would prove the efficacy of Hok-Sok as an alternative plasmid maintenance system and method for combating the widespread use of antibiotics and growing population of antibiotic resistant bacteria.</p>
+<p style = "font-size:18px">As mentioned earlier, many synthetic biology projects, including those within iGEM, intend to release their final product into nature. As our testing supports the conclusion that Hok-Sok can successfully be implemented to maintain plasmids without antibiotics, it would be intriguing to test this further ourselves or see other iGEM teams test this by implementing the Hok-Sok cassette as the maintenance system on a project where the final product is intended for release into  nature. Successful implementation of this would further prove the efficacy of Hok-Sok as an alternative plasmid maintenance system and as a method for combating the widespread use of antibiotics and growing population of antibiotic resistant bacteria.</p>
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