Purpose

Exploring the situation of oral environment and send out warning signal if problems concerning tooth decay are going to emerge. With the banana fragrance getting stronger and stronger, people will be able to know that the risk of cavity in their mouth is on the rise.

Background

The special organism we adapted in this part is Streptococcus sobrinus. By using its specific biological feature, we can turn our circuit into an alarm clock, which would ring whenever humans are in danger of oral cavities. Most people don’t know about the existence of cavities in their mouths until suffering from toothaches or serious pain. However, it is difficult for people to go for dentist checks very often because of the high expenses and amount of time spent. Furthermore, for people in rural areas, there might be insufficient dental resources. Therefore, we have decided to create a system that can alert people from time to time. We aim to use banana fragrance as our indicating method instead of visual cues, avoiding the embarrassment that might result from unsightly colors appearing on our tooth.

Chassis

For this circuit, we use a special chassis to load and express our wanting genes, which is Streptococcus sobrinus, it is an anaerobic, spherical shaped, Gram positive bacteria. The reason why Streptococcus sobrinus can serve as a good indicator is that the group scale of Streptococcus sobrinus would change dramatically when tooth decay is about to happen. Therefore, when there are lots of modified Streptococcus sobrinus in our oral environment, they will release strong banana odor to alert people to see the dentist.

Design

Promoter (iGEM part BBa_J23100)

It is a commonly used constitutive promoter of iGEM part registry.

RBS (iGEM part BBa_B0030)

It is a commonly used ribosome binding site of iGEM part registry.

The banana odor generator consists of 3 parts.

Part 1 (iGEM part BBa_J45008 [1,2,5])

BBa_J45008 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, BAT2 gene. The function of this part is to produce enzyme to get involved in the first step in the aging reaction of L-leucine to isoamyl alcohol. We got this part from the 2007 iGEM kit.

Part 2 (iGEM part BBa_J45009 [1,3,6])

BBa_J45009 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, THI3 gene. The function of this part is to produce enzyme to get involved in the second step in the aging reaction from L-leucine to isoamyl alcohol. Because this part is not released, we cultured Saccharomyces cerevisiae S288c and extract the DNA of it.

Part 3 (iGEM part BBa_J45014 [1,4,7])

BBa_J45014 is first used by 2006 MIT iGEM team, and originated from Saccharomyces cerevisiae S288c, ATF1 gene. The function of this part is to produce enzyme to get involved in the esterification reaction from isoamyl alcohol to isoamyl acetate, which brings out the banana flavor. We got this part from the 2014 iGEM kit.

Terminator (iGEM part BBa_B0015)

A double terminator composed of B0010 and B0012. It has used by many iGEM teams, and have strong terminating force.

Functional Measurement

Circuits needed to be constructed

Promoter (BBa_J23100) + RBS (BBa_B0030) + Banana odor generator ( BBa_J45008+BBa_J45009 +BBa_J45014)+Terminator(BBa_B0015)

Experimental steps

1. Culture modified E. coli in sealed agar plates overnight.
2. Use syringe to extract the gas generated by E. coli.
3. Use gas chromatography to analyze the sample.

Result

Reference

1. 2006 MIT iGEM team and iGEM part registry BBa_J45008 / BBa_J45009 / BBa_J45014
2. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome X, complete sequence
3. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome IV, complete sequence
4. NCBI Blast: TPA_inf: Saccharomyces cerevisiae S288c chromosome XV, complete sequence
5. (2012)"Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis"