Difference between revisions of "Team:Qdai/Engineering"

DBTL(Design-Build-Test-Learn) cycle

We adopted the DBTL cycle and tried to make it reproducible by others by showing the basic parts and composite parts used and the lab notebook.

1. Design

We have designed a bacterium that can detect carbon monoxide. We introduce the carbon monoxide sensor protein CooA and the photoprotein luciferase into E. coli.CooA is a carbon monoxide-dependent transcriptional activator. CooA contains heme and activates CooA-dependent promoters when it binds CO. Firefly luciferase is an enzyme that catalyzes a bioluminescent reaction in the presence of Mg2+. We expect that the luciferase produced by E. coli and D-luciferin added to the culture medium will cause the following reaction.

luciferase+luciferin+ATP↔luciferase∙luciferyl‐AMP+PPi
luciferase∙luciferyl‐AMP+O2→luciferase+oxyluciferin+AMP+CO2+hv

CooA, which is always produced when CO is nearby, binds to CO. pCooM has the gene for luciferase downstream, so we can add luciferin to the medium to output light. Since our E. coli is used as a CO detector, we need to adjust the CO sensitivity and make it emit light at the optimal CO concentration. We believe this can be achieved by using promoters with different strengths and RBS.For example, by combining two CooA-dependent promoters of different strengths and two RBSs of different strengths, we can construct four different patterns.Details can be found in the methodology.

2. Build

Modeling was used to predict the relationship between carbon monoxide concentration and luciferase expression.

From the above reaction equation, the rate reaction equation for the formation of CooAco is as follows.

Here, the concentration of luciferase is expressed by the following equation

Assuming that t is very large, this equation leads to the following graph. This graph predicts the relationship between CooAco and luciferase expression levels at steady state.

CooA continues to be transcribed, but the amount of protein will eventually reach a steady state. After a sufficient amount of time, the amount of CooAco can be considered to depend on the CO concentration, and the following equation can be obtained. The following graph can then be obtained.

3.Test

3. Test Next, we predicted that there would be a negative effect on growth based on the effect of the CooA gene being expressed normally and the effect of anaerobic conditions as the CO concentration increased. We thought that we could predict just the right balance between logistically suppressed growth and light intensity.

We considered the negative effect of CO on growth at f, and the negative effect of the production of proteins that are not originally produced by E. coli at g. Here is the equation.

Z was defined as follows.

Z represents the amount of light and how responsive it is as a sensor.

Q=NZ

Q represents the ability of the E. coli aggregate to act as a sensor and the degree of luminescence. More details can be found on the modeling page.

While we were actually conducting experiments in the laboratory, we faced the problem that it was difficult to see the luminescence reaction that occurs in E. coli with the naked eye. From there, a new method of using nanolanterns that can be seen with the naked eye can be considered. In our experiments, we thought the ligation was not good because the vector did not contain the insert, so we decided to purify the DNA after the Gibson assembly.
Still, the insert didn't fit, so we changed both ends of the insert so that it was all assembled by Gibson.In addition, we still didn't work, so we decided to split Gibson in two parts. Especially in the experiment we repeated a small cycle. More details can be found in the notebook.

4. Learn

We discussed with Hiroto Tanaka of the National Institute of Information and Communications Technology (NICT), Nagaura farm, Micro bio factory Inc, Figaro Engineering. The discussions with people from various backgrounds gave us a multifaceted perspective. For example, the discussion with Figaro Engineering gave us a hint on how to make the CO detection system work practically. We also went to a cattle barn to observe the actual site where the results would be used.