Team:NCKU Tainan/Improve
Improve
Part Improvement
During the designation of the experiment for our pH alert system, we have concluded that we should try to improve the expression of our pH-sensitive promoter, promoter gadA. The motivation for us to do this is because our team think that if we can build a system which could observe the situation in our device by looking at the color change of the medium, it should be an economic friendly as there is no more extra charges to send your sample to someway for checking and also wasting a lot of time for waiting the result. But according to the expression of promoter gadA from the previous team did, it is not enough to be observed by using our eyes.
Based on our research, RiboJ first described as it is an insulator in genetic construct but there has no any data shown this insulation effected the downstream genetic parts. In 2018, there is a team have proved that RiboJ could increase the expression of downstream gene and there is the team member, Kalen P Clifton#1, Ethan M Jones#1, Sudip Paudel1, John P Marken2, Callan E Monette1, Andrew D Halleran2, Lidia Epp1, Margaret S Saha1*. Hence, our team, NCKU_Tainan have desided to enhance the expression and specificity of promoter gadA by adding a gene, RiboJ at the downstream of promoter gadA.
For the function determination, we have measured the fluorescent density of the plasmid with and without RiboJ in different pH environment. We incubated the bacteria in pH adjacent M9 medium and measure the fluorescent intensity (absorbance: 480nm, excitation: 510 nm) in a short period of time.
Experiment Result
This is the third year since the establishment of iGEM NCKU-Tainan team. We have raised the awareness of synthetic biology in our campus. Mrs. Jenny Su and the College of Engineering decided to give us a headquarter which contains a meeting room and a lab. To meet the lab standard of CDC (Center for Disease Control), we made contact with Mrs. Liu Yee Fen, the contact person of Center for Occupational safety and Health and Environmental Protection, which is the authorized unit in our campus responsible for lab safety and security. With the assistance of our instructor Dr. Ng, we successfully certify our lab to BSL1 saftey level one lab. Mrs. Liu gave us some lab security instructions and advice. She also suggested that each of our members should attend the biosafety lab training program.
Expect the lab in iGEM headquarter, wet team has constructed out design in various labs whose supervisors are Dr. I-Son Ng, Dr. I-Hsiu Huang, Dr. Han-Ching Wang, and Dr. Hashimoto Masayuki. The labs mentioned above are all certified biosafety level one.
Experiment Result
Figure 1: The fluorescent intensity expressed by PgadA of 24 hours in four different pH value of M9 medium.
Figure 2: The fluorescent intensity expressed by PgadA-RiboJ of 24 hours in four different pH value of M9 medium.
＆nbsp＆nbsp＆nbsp＆nbsp＆nbspFigure 3: The fluorescent intensity between PgadA and PgadA-RiboJ in pH6 and pH 7.
Experiment Result
Based on our functional determination test results, the improvement has succeeded as RiboJ really could enhance the expression and specificity of downstream gene.