Team:SDSZ China/Pa
Basic Part 1: BBa_I712019 (luciferase)
http://parts.igem.org/Part:BBa_I712019
Aim
We developed a new usage of luciferase – detecting bacteriophage density in fecal polluted water. When different phage densities (1x, 2x, 5x, 10x ideally) were prepared and substrate solution (D-luciferin, MgSO4, ATP) was added to the liquid LB culture of E.coli. solution, cell lysis occurred and the expressed
Measurements and Reactions
1. ModelWe have documented the OD values when conducting IPTG induction to get the best level of protein expression. As induction proceeds and OD600 value reaches somewhat near 0.3, protein expression is tested to be at high level by mathematical model.
2. Solution and reactionThrough various tests and professional journals, we have prepared effective substrate solution for our detection system listed as below:
Mg2+ 0.001mol/L ATP 0.001mol/L D-luciferin 5mg E.coli. solution with OD value around 0.3 500ul Add liquid LB medium to the whole reaction system until volume reaches 3000ul.
Under this substrate system, the reaction went on smoothly when bacteriophages were introduced to the solution.
3. ResultsAs we added phage solutions to the culture medium, bioluminescence was observed and recorded as below.
-From left to right: negative control | 2x | 5x
figure under natural light | figure under ultraviolet light
Basic Part 2: BBa_K3091000 (Polyphenol Oxidase)
http://parts.igem.org/Part:BBa_K3091000
Aim
We built this new part as an approach to detecting bacteriophages in fecal polluted water through color change. This enzyme is from Solanum tuberosum, and would cause yellow solution containing tea polyphenol turn red. Also, the product of the reaction, theaflavins, has multiple health benefits. Our bacteria can potentially also play a part in theaflavins industrial production.
Measurement and Reaction
1. ModelWe have documented the OD values when conducting IPTG induction to get the best level of protein expression. As induction proceeds and OD600 value reaches somewhat near 0.3, protein expression is tested to be at high level by mathematical model.
2. Solution and ReactionThrough various journals, we learned that potato PPO is most active at PH=5.5~7, temperature about 30~40℃. As for the substrate concentration, we still need to conduct control experiments because tea polyphenol can potentially have anti-bacteria effects. More tests should be done to find an ideal solution that make the reaction visible and reliable.