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Control-Sensor

Purpose

We are going to use the “control part” to control the number of Streptococcus mutans (S. mutans). When the number of S. mutans exceeds the threshold that causes cavities, the circuit will be activated, thus killing the excess S.mutans.

Background

It is said that the number of S. mutans is highly related to the outbreak of cavities. Therefore, controlling the number of S. mutans has been our first priority. To reach our purpose, we need to detect the number of S. mutans, determine the threshold that causes cavities, and finally, kill the S. mutans. These three main designs of our first C, “control”, will be explained in the following paragraphs.

First of all, we need to detect the number of S. mutans. Over the years, it has been found that the competence stimulating peptide, CSP, a quorum sensing chemical, will be released in every competence S. mutans. Thus, we can detect the number of S.mutans by marking the amount of CSP.

In S.mutans, the CSP will bind to the membrane receptor, “comD”, thereby phosphorylating the response regulator, “comE”. The phosphorylated comE will activate numerous vital biological mechanisms in Streptococcus mutans such as biofilm formation and mutacin release. Compared with all the other mechanisms involved with CSP, it is found that the promoter of gene “nlmC”( non-lantibiotic mutacin C) in S.mutans has the best performance under the stimulation of CSP. As a result, we have decided to use this promoter in our design.

Secondly, we need to find and confirm the threshold that causes cavities. In this part, we will use different kinds and numbers of terminators (each with different leakage rates) to create the threshold. Moreover, we will combine both wet lab results and modelling to decide which design is more suitable.

Lastly, we need to kill the S. mutans when the amount of S. mutans exceeds the threshold. To fulfill our goal, we decided to incorporate our circuit into the Streptococcus phage M102, which is highly specific to S. mutans, in order to precisely control the lysis genes in phage M102.

Design

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In our circuit, we incorporate nlmC promoter and different combination of terminators. When the number of S. mutans exceed the threshold, the first “C” , control mechanism will activate the lysis genes of phage M102. Thus, control the number of S. mutans.

In order to test the function of the “control” part of this project, we incorporate two stages of functional tests.

The first stage
We will construct two circuits. One is served as a positive control, constructed with a constitutive promoter and the GFP gene from the iGEM kit, E0040. The other is served as an experimental group, constructed with a nlmC promoter and E0040. When doing the functional measurement, we will apply different amount of synthetic CSP in different time lapse into our experimental group after the circuit being transformed into S. mutans.
In the first stage of functional test, it is hoped to see the high amount of green fluorescent in the positive control, while different amount of green fluorescent being observed in our experimental group.

The second stage
We will test the circuit constructed with nlmC promoter and different kinds of terminator, also using E0040 to measure the threshold we create with terminators. In this stage, we will test three different terminators, B0012, B0015, and one derived from S.mutans in different combination, namely, we will construct nine circuits:

• nlmC promoter + B0012 + E0040
• nlmC promoter + B0015 + E0040
• nlmC promoter + terminator from S.mutans + E0040
• nlmC promoter + B0012 + B0012 + E0040
• nlmC promoter + B0012 + B0015 + E0040
• nlmC promoter + B0012 + terminator from S.mutans + E0040
• nlmC promoter + B0015 + B0015 + E0040
• nlmC promoter + B0015 + terminator from S.mutans + E0040
• nlmC promoter + terminator from S.mutans+ terminator from S.mutans +E0040

In the second stage of our functional test, we are looking forward to see different amount of green fluorescent after applying different amount of synthetic CSP in different time lapse into these nine circuits.
After the second stage of the function test, we will hand over our data to our modeling group, to see which threshold suit best in killing S.mutans.

Result

Reference

1. Kreth, J., Hung, D. C. I., Merritt, J., Perry, J., Zhu, L., Goodman, S. D., Cvitkovitch, D. G., ... Qi, F. (January 01, 2007). The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis. Microbiology Reading-, 153, 1799-1807.
2. Hung, D. C. I., Downey, J. S., Ayala, E. A., Kreth, J., Mair, R., Senadheera, D. B., Qi, F., ... Goodman, S. D. (June 28, 2011). Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans. Journal of Bacteriology, 193, 14, 3642-3652.
3. Liu, T., Xue, S., Cai, W., Liu, X., Liu, X., Zheng, R., Luo, H., ... Qi, W. (March 22, 2014). ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans. Annals of Microbiology, 64, 1, 31-38.
4. van der Ploeg, J R. (2007). Genome sequence of Streptococcus mutans bacteriophage M102. Wiley-Blackwell.

Control-Inhibitor

Purpose

Our inhibitor part aims to decrease the intrinsic ability of biofilm formation of S. mutans. In the human mouth, S. mutans tends to produce biofilm and acid product through metabolism. Biofilm provides a comfortable environment for S .mutans to survive and live in. Because of this, we try to decrease the formation of biofilm by S.mutans in order to decrease the chance of tooth decay.

Background

Histidine kinase is a sensor kinase of two-component signal transduction system. According to literature search, deletion of histidine kinase will result in biofilm formation and resistance to acidic pH. Scanning electron microscopy also show that S. mutans forms sponge-like biofilm.

G protein in S. mutans(SGP) is involved in regulating the intracellular GTP/GDP ratio, response to stress condition, and other diverse cellular functions. Based on our paper search, deletion of SGP also showed that biofilm formation decreased.

Design

We synthesized a 24 bp non-coding DNA and transcribed it into sRNA. This short sRNA will bind to the TIR (translation initiation region) of target mRNA and prevent the target mRNA from translating. We target two biofilm formation-related protein: one is histidine kinase and the other is G protein. According to literature search, defection of these two protein will dramatically decrease the biofilm formation of S. mutans. Another important feature is the MicC scaffold, which will recruit Hfq protein and help sRNA hybridize with target mRNA, while also stabilizing the sRNA-mRNA complex.

Result

Reference