Biosafety

Organisms We Used

We use E.coli DH5α and phage M102 in our project. They help us to control the number of S. mutans, pathogen in the mouth. As we hope to use these organisms inside our mouth, we do biosafety research, and design suicide circuits in E. coli to prevent it from causing disease when dropping out of our mouth. We will discuss biosafety, laboratory safety respectively.

Strain information of E. coli

E. coli DH5α is in risk group 1, which is the most harmless. They do not carry the well recognized pathogenic mechanisms required by strains of E. coli that cause the majority of enteric infections. It is considered to be non‐pathogenic and unlikely to survive in host tissues and cause disease. [1]

Strain information of bacteriophage M102

Phage M102 belongs to the family of Siphoviridae with morphotype B1[2], which is tail long and noncontractile, head isometric. Bacteriophage M102 is specific to S. mutans, there is little concern of risks to human and environment.

Purpose

We use Lactobacillus Casei as our chassis in bringing out numeral functions. However, we use E.coli primarily to do our circuit construction, and the testing of its features in biofilm-cleansing and pathogen killing. It is obvious that Lactobacillus Casei has less safety concern in contrast to E.coli. Therefore, in afraid of causing environmental danger, we design several safety lock, in the aim of containing harms.

Design

We constructed a killing gene regulated by a blue light promoter. We picked Ccdb, which is a coding gene that blocks E.coli polymerase to bind on DNA. As for the blue light promoter we uses FixK2 promoter which is regulated by blue light. Both these gene were separately K145151 and K592006 in the iGEM part registry.

Experiment

Six plates of biosafety E.coli was cultured in individual 10 microliter LB. Two in a group one test one control. Therefore, we have three groups of data (6 Control / 6 Test ) , (12 Control / 12 Test ) , (24 Control / 24 Test ) All six of them are covered with Aluminum foil, in order to isolate blue light disturbance.

After the culture reaches 6, 12 and 24 hour, we open the aluminum foli of the test group. We plotted the surviving cell number over blue light exposure time by doing CFU (colony Forming Unit), and hence we derive a surviving ratio over light exposure time.

Result

Due to the killing efficiency attained less than 1 survival in every 100 population at the growing time 6 hour. The CFU counting may not be a suitable alternative. Therefore, we did rough estimation by a serial 10X times dilution. By setting the gradient at 10 times dilution, we can know where the cell number’s power index lye. For example, the colony disappear between gradient of 4 and 5, thus we can infer the cell number X would be : 10⁴ < X < 10⁵ . So on and on, there are several examples in graph.

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Lab safety

We conducted our experiments in Biosafety level 1 laboratory, which is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment according to CDC(Centers for Disease Control and Prevention in United states). Furthermore, professors checked our experiments before we did it. And we have written the protocols to confirm that we had right procedures.

Lab training

Every person in NYMU iGEM team has 3 in 1 training directed by professors, who led us step by step. Thus, some toxic chemicals are emphasized, like DNA dye used in electrophoresis. After doing all steps overseen by professors, all people could use instruments properly.

Protection

Everyone must wear coat and gloves before entering the laboratory with long pants and shoes which covered our feet entirely. If we need to go out of the experimental zone, we take off the coat and gloves, and washed hands with antibacterial hand wash, not to touch door knob with gloves. We have done this strictly, so to prevent taking the bacteria out of lab.

Waste

There is chemical and bacteria mixture after conducting the experiment, we gather then into a bottle. Clean it daily to prevent explosion because of air made by bacteria. After autoclaving the waste, we pour it with a lot of water to dilute the toxic chemicals. Instead of carcinogen EtBr, we used safer dyes to reduce contamination.

Reference

1. Escherichia coli K-12 Derivatives Final Risk Assessment , TSCA (http://epa.gov/biotech_rule/pubs/fra/fra004.htm)
2. van der Ploeg, J. R. (2007). Genome sequence of Streptococcus mutans bacteriophage M102. FEMS Microbiol Lett 275, 130–138.