Team:Pitt/Protocol Design/Results

From 2014.igem.org

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.