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Team:Pitt/Protocol Design/Results
From 2014.igem.org
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| - | <h2 id = "results">Results</h2> | + | <h2 id = "results">Protocol Results</h2> |
<p>As of 10/16/2014, 12 of 16 trials have currently been completed with no positive results (Table 1). The largest hurdle to completing trails was the fact that 4 of the strains that were grown at 24°C did not ever reach the optical density that was required for an attempt at transformation. In addition, the other 4 strains grown at 24°C that did reach an O/D of around 0.8 took much longer than the 2 weeks that it took for the strains grown in the 37°C room. Since, we did not have 16 trails, we could not perform a DOX analysis to determine which variables were the most important for transformation. This led us to conclude that if another DOX analysis is conducted with these parameters, it would be efficacious to use a low value for culture temperature that is higher than 24°C in order to at least have viable <i>P. Acnes</i> to perform a transformation protocol with.</p> | <p>As of 10/16/2014, 12 of 16 trials have currently been completed with no positive results (Table 1). The largest hurdle to completing trails was the fact that 4 of the strains that were grown at 24°C did not ever reach the optical density that was required for an attempt at transformation. In addition, the other 4 strains grown at 24°C that did reach an O/D of around 0.8 took much longer than the 2 weeks that it took for the strains grown in the 37°C room. Since, we did not have 16 trails, we could not perform a DOX analysis to determine which variables were the most important for transformation. This led us to conclude that if another DOX analysis is conducted with these parameters, it would be efficacious to use a low value for culture temperature that is higher than 24°C in order to at least have viable <i>P. Acnes</i> to perform a transformation protocol with.</p> | ||
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<p>The goal of our part of the experiment was to develop a transformation protocol for a bacteria that was on the skin. This would allow us to incorporate a plasmid into the bacteria that could produce a protein to reduce acne. <i>P. Acnes</i> was the obvious choice since it is abundant on the skin and aggressive strains of the bacteria are associated with acne formation. As we discovered, however, it is rather difficult to transform. This led us to consider other bacteria on the skin that are easier to transform.</p> | <p>The goal of our part of the experiment was to develop a transformation protocol for a bacteria that was on the skin. This would allow us to incorporate a plasmid into the bacteria that could produce a protein to reduce acne. <i>P. Acnes</i> was the obvious choice since it is abundant on the skin and aggressive strains of the bacteria are associated with acne formation. As we discovered, however, it is rather difficult to transform. This led us to consider other bacteria on the skin that are easier to transform.</p> | ||
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<tr><td style = "font-size:14"><b>Name of Bacteria</b></td><td style = "font-size:14"><b>Skin Locations</b></td> <td><b>Transformation Protocols found on the web</b></td><td style = "font-size:14"><b>Notes</b></td></tr> | <tr><td style = "font-size:14"><b>Name of Bacteria</b></td><td style = "font-size:14"><b>Skin Locations</b></td> <td><b>Transformation Protocols found on the web</b></td><td style = "font-size:14"><b>Notes</b></td></tr> | ||
| - | <tr><b><i style = "background-color:green">Phylum:</b> Firmicutes<br> | + | <tr><td><b><i style = "background-color:green">Phylum:</b> Firmicutes<br> |
<b>Species: Staphylococcus aureus</b></i></td> | <b>Species: Staphylococcus aureus</b></i></td> | ||
<td><b>Relevant locations:</b> | <td><b>Relevant locations:</b> | ||
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<td><b>Potential Safety concern:</b> Can be pathogenic</td></tr> | <td><b>Potential Safety concern:</b> Can be pathogenic</td></tr> | ||
<tr> | <tr> | ||
| - | <td><i><b>Phylum: Firmicutes</b><br> | + | <td><i style = "background-color:green"><b>Phylum: Firmicutes</b><br> |
<b>Species:</b> Streptococcus pyogenes</i></td> | <b>Species:</b> Streptococcus pyogenes</i></td> | ||
<td><b>Relevant locations:</b> | <td><b>Relevant locations:</b> | ||
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<td><a href = "http://www.sciencedirect.com/science/article/pii/0378109791903369">http://www.sciencedirect.com/science/article/<br>pii/0378109791903369</a></td> | <td><a href = "http://www.sciencedirect.com/science/article/pii/0378109791903369">http://www.sciencedirect.com/science/article/<br>pii/0378109791903369</a></td> | ||
<td><b>Potential Safety concern:</b> Can be pathogenic</td></tr> | <td><b>Potential Safety concern:</b> Can be pathogenic</td></tr> | ||
| - | <tr><td><i><b>Phylum:</b> Actinobacteria<br> | + | <tr><td><i style = "background-color:yellow"><b>Phylum:</b> Actinobacteria<br> |
<b>Genus:</b> Corynebacteria<br> | <b>Genus:</b> Corynebacteria<br> | ||
<b>Species:</b> C. amicolatum, C. striatum, C. jeikeium, C. urealyticum, and C. xerosis</i></td> | <b>Species:</b> C. amicolatum, C. striatum, C. jeikeium, C. urealyticum, and C. xerosis</i></td> | ||
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<td>None found</td></tr> | <td>None found</td></tr> | ||
<tr> | <tr> | ||
| - | <td><i><b>Phylum:</b> Actinobacteria<br> | + | <td><i style = "background-color:green"><b>Phylum:</b> Actinobacteria<br> |
<b>Family:</b> Micrococciaceae<br> | <b>Family:</b> Micrococciaceae<br> | ||
<b>Species:</b> M. lysodeikticus</i></td> | <b>Species:</b> M. lysodeikticus</i></td> | ||
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<td>Potential Safety concern: Can be pathogenic</td></tr> | <td>Potential Safety concern: Can be pathogenic</td></tr> | ||
<tr> | <tr> | ||
| - | <td><i><b>Phylum:</b> Actinobacteria<br> | + | <td><i style = "background-color:yellow"><b>Phylum:</b> Actinobacteria<br> |
<b>Genus:</b> Propionibacteriaceae<br> | <b>Genus:</b> Propionibacteriaceae<br> | ||
<b>Species:</b> P. acnes, P. acidifaciens, P. australiense, P. avidum, P. granulosum, P. humerusii, P. lymphophilum, P. propionicum</td> | <b>Species:</b> P. acnes, P. acidifaciens, P. australiense, P. avidum, P. granulosum, P. humerusii, P. lymphophilum, P. propionicum</td> | ||
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</table> | </table> | ||
</center> | </center> | ||
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<hr> | <hr> | ||
<h2 id = "conclusions">Conclusions</h2> | <h2 id = "conclusions">Conclusions</h2> | ||
| - | + | <center> | |
| - | < | + | <img src = "https://static.igem.org/mediawiki/2014/thumb/b/bd/Pitt_dox_results1.png/418px-Pitt_dox_results1.png"> |
| - | <p> | + | <p>From: “The Skin Microbiome.” Grice 2011.</p> |
| - | < | + | </center> |
| - | + | <p>Unfortunately, our experiments did not provide any positive results. However, future experiment should attempt to use different bacteria and also higher temperatures for culture. In addition, the plating of the bacteria should be done with utmost care, near a flame to prevent contamination. </p> | |
| + | <br> | ||
<a href = "https://2014.igem.org/Team:Pitt/HSP60_Promoter/Intro"> | <a href = "https://2014.igem.org/Team:Pitt/HSP60_Promoter/Intro"> | ||
<div class = "next_page"> | <div class = "next_page"> | ||
Latest revision as of 19:21, 17 October 2014
Protocol Results
As of 10/16/2014, 12 of 16 trials have currently been completed with no positive results (Table 1). The largest hurdle to completing trails was the fact that 4 of the strains that were grown at 24°C did not ever reach the optical density that was required for an attempt at transformation. In addition, the other 4 strains grown at 24°C that did reach an O/D of around 0.8 took much longer than the 2 weeks that it took for the strains grown in the 37°C room. Since, we did not have 16 trails, we could not perform a DOX analysis to determine which variables were the most important for transformation. This led us to conclude that if another DOX analysis is conducted with these parameters, it would be efficacious to use a low value for culture temperature that is higher than 24°C in order to at least have viable P. Acnes to perform a transformation protocol with.
Table 1
| Date: | Std/Run | Results: |
| 8/8/14 | 11/16 | Lawn, phage test showed it was not p. acnes |
| 8/8/14 | 15/3 | 17 colonies, colony PCR showed that they didn’t contain the plasmid |
| 8/8/14 | 3/11 | 73 colonies, colony PCR showed that they didn’t contain the plasmid |
| 9/2/14 | 9/10 | No growth after electroporation |
| 9/2/14 | 5/9 | No growth after electroporation |
| 9/2/14 | 1/2 | No growth after electroporation |
| 9/23/14 | 4/5 | 3 colonies, not p. acnes by color and growth pattern |
| 9/23/14 | 6/13 | No growth after electroporation |
| 9/23/14 | 7/6 | 1 colony, not p. acnes by color and growth pattern |
| 9/23/14 | 13/15 | No growth after electroporation |
| 9/23/14 | 10/8 | No growth after electroporation |
| 9/23/14 | 16/14 | No growth after electroporation |
The goal of our part of the experiment was to develop a transformation protocol for a bacteria that was on the skin. This would allow us to incorporate a plasmid into the bacteria that could produce a protein to reduce acne. P. Acnes was the obvious choice since it is abundant on the skin and aggressive strains of the bacteria are associated with acne formation. As we discovered, however, it is rather difficult to transform. This led us to consider other bacteria on the skin that are easier to transform.
We came up with a number of candidates for transformation and identified transformation protocols. The information below indicates which bacteria are the best for transformation:
Top candidates ideal skin location and transformation protocol available: S. aureus, S. epidermidis, M. lysodeikticus.
Top candidates ideal skin location but no transformation protocols found by search: P. acnes, P. acidifaciens, P. australiense, P. avidum, P. granulosum, P. humerusii, P. lymphophilum, P. propionicum, C. amicolatum, C. striatum, C. jeikeium, C. urealyticum, and C. xerosis.
| Name of Bacteria | Skin Locations | Transformation Protocols found on the web | Notes |
| Phylum: Firmicutes Species: Staphylococcus aureus |
Relevant locations:
Occiput, Glabella, Manubrium Other locations: Plantar Heel, Popiteal fossa, Anticubital fossa, Toe web space | http://lucigen.com/docs/posters/High-efficiency-direct-transformation-of-Staphylococcus-aureus-2013.pdf http://www.dartmouth.edu/~staphy/protocol/transformation.html http://mbio.asm.org/content/3/2/e00277-11.full.pdf |
Potential Safety concern: Can be pathogenic |
| Phylum: Firmicutes Species: Streptococcus pyogenes |
Relevant locations:
None Other locations: Gluteal crease |
http://www.sciencedirect.com/science/article/ pii/0378109791903369 |
Potential Safety concern: Can be pathogenic |
| Phylum: Actinobacteria Genus: Corynebacteria Species: C. amicolatum, C. striatum, C. jeikeium, C. urealyticum, and C. xerosis |
Relevant locations:
Occiput, Glabella, Manubrium, back Other: external auditory canal, nare, inguinal crease, umbilicus, toe web space |
No efficient transformation protocol for listed species but some available for C. Glutamicum | |
| Phylum: Firmicutes Species: Staphylococcus epidermidis |
Relevant locations:
Occiput, Glabella, Manubrium Other: Plantar Heel, Popiteal fossa, Anticubital fossa, Toe web space retroauricular crease, occiput, gluteal crease | http://www.sciencedirect.com/science/article/ pii/037810979090283V http://mbio.asm.org/content/3/2/e00277-11.full.pdf | |
| Phylum: Proteobacteria Class: Betaproteobacteria Species: ?, |
Relevant locations:
Occiput, back Other: Axillary vault, Antecubital fossa, volar forearm, hypothenar palm, interdigital web space, buttock, plantar heel |
None found | |
| Phylum: Bacteriodetes Species: ? |
Relevant locations:
Occiput, back Other: Axillary vault, antecubital fossa, volar forearm, hypothenar palm, interdigital web space, buttock |
None found | |
| Phylum: Actinobacteria Family: Micrococciaceae Species: M. lysodeikticus |
Relevant locations:
Occiput, back Other: Toe web space |
http://jb.asm.org/content/98/3/1397.full.pdf | Potential Safety concern: Can be pathogenic |
| Phylum: Actinobacteria Genus: Propionibacteriaceae Species: P. acnes, P. acidifaciens, P. australiense, P. avidum, P. granulosum, P. humerusii, P. lymphophilum, P. propionicum |
Relevant locations:
Glabella, Manubrium, back Other: Alar crease, external auditory, retroauricular crease, buttok, gluteal crease |
Some for P. acnes found in literature but not able to replicate | Potential Safety concern: Can be pathogenic |
Conclusions
From: “The Skin Microbiome.” Grice 2011.
Unfortunately, our experiments did not provide any positive results. However, future experiment should attempt to use different bacteria and also higher temperatures for culture. In addition, the plating of the bacteria should be done with utmost care, near a flame to prevent contamination.
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