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 enzymes would release to react with their substrate to emit bioluminescence.

Plasmid Construction

Because we didn’ t receive the distribution kit when our experiment began, we acquired the Photinus pyralis Luciferase sequence from a vector purchased from addgene (luciferase vector addgene-plasmid-37851-sequence-180888). Our sequencing results showed that the sequence of our luciferase DNA fragment is identical to part BBa_I712019 in the registry.

We selected pET expression system for its high efficiency and capability to be regulated by IPTG induction.

We constructed the plasmid using digestion and ligation. After failures and testing reasons of failures and improvements, we finally constructed the plasmid successfully. Then we used IPTG to induce protein expression.

Measurements and Reactions

1. Model

We 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.

Note:the OD values to get the best level of protein expression are determined by analyzing the data and standard transformations.

2. Solution and reaction

Through 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. Results

As we added phage solutions to the culture medium, bioluminescence was observed and recorded as below.

figure under natural light | figure under ultraviolet light

-From left to right:

negative control | 5x coliphage solution | 2x coliphage solution

Note: Since there is no proper device in our lab for observing bioluminescence, the observation and documentation of our detection result went on rather difficultly. Although D-luciferin may seem to be faintly luminescence under ultraviolet light, we confirmed that it does not emit any observable luminescence in complete darkness.

Basic Part 2: BBa_K3091000 (Polyphenol Oxidase)

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.

Plasmid Construction

We acquired the gene sequence through NCBI blast, and synthesized the sequence thanks to Twist, and selected pET expression system due to its high efficiency and capability to be regulated by IPTG induction.

We tried to use digestion and ligation to construct the plasmid, but it failed numerous times. We speculate the reason is that the efficiency of the enzyme Bgl2 is lowered due to long time storage in the lab. Then we borrowed in-fusion mix from BNU and built the plasmid again, and finally succeed.

Measurement and Reaction

1. Model

We 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 Reaction

Through various journals, we learned that potato PPO is most active at PH=5.5, temperature about 40~50°C. 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.

Team:SDSZ China/Contribute

Overview:

Basic Part 1: BBa_I712019 (luciferase)

Basic Part 2: BBa_K3091000 (Polyphenol Oxidase)