Team:WHU-China/Proof Of Concept
Overview
To develop a novel acne therapy, we engineered a safe, nutritionally deficient Escherichia coli strain, which could decompose fatty acids in high efficiency(BBa_K3763044, BBa_K3763045 ) and secrete strain-specific bacteriocin( BBa_K3763023, BBa_K3763024), for ameliorating sebum blocking and inhibiting P. acnes growth, respectively. In addition, a fatty acid-sensing system (BBa_K3763001, BBa_K3763002, BBa_K3763003) was constructed to enable precise therapeutics actuation only in appropriate conditions, of which we designed a directed evolution pipeline to further optimize the function.
Through a year’s preparation and lab work, we’ve achieved basic proof of concept via several fundamental experiments, hardware and models. We will demonstrate that our designs are able to play a role in the following content.
Fatty acid sensing module
In this part, we first tested the function of the fatty acid-sensitive promoter pFadD_Lac, on which we set the basis of our design logically. The result showed that both the original and the modified promoter-RBS combination responded to changes in fatty acid concentrations, indicating that this fatty acid sensing system did function as we expected. And we have successfully improved the performance of the RBS, making our engineered bacteria more sensitive to fatty acids.
Then for the directed evolution, we carried out epPCR experiment and sequenced the PCR product. The result showed that overlapping peaks occurred in the epPCR product, indicating that we successfully introduced mutations into the sequence of interest, and thus provedthe possibility of constructing a mutation library with the potence of providing the sequence we need.
Fatty Acid Consumption Module
In this part, we carried out experiments to test how the overexpression of the β-oxidation related enzymes affected fatty acid consumption. Although the result showed that there was no distinct difference between the wild type and the FadE/FadR overexpression strain in the speed of consummating fatty acids, there was still a growing tendency for FadE/FadR overexpression strain that seemed prosperous. As figure 3 shows, there is a positive correlation between the induction time and the consumption of overexpression strain, which means that the two enzymes were functioning as we expected.
Since we highly speculated that the overexpressed FadE and FadR were easy to be degraded and thus didn’t bring the engineered bacteria the character we intended, we have changed chassis into BL21 and carried out new experiments. We believe that after the change of chassis we will have a result strongly supporting our design.
Hardware
For the hardware, we have accomplished the systematical work of turning it from design into reality. The results showed that the microfluidic chip has successfully passed the tests of chip matching, chip path detection, loading beads and bacteria and finally form water-in oil droplets. It has been proved that in high-throughput arrays, bacteria that have the target gene required for directed evolution will be captured, enabling us to use a single cell as the templet to obtain a mutation library of FadR by error-prone PCR.
For the Inhibition Module and Safety Module, we are half way towards success. Please visit engineering for more details.
Model
The enzymes we use in Fatty Acid Consumption Module is selected by flux accelerator model which have the most potential to enhance consumption of higher fatty acids.
In addition, although we haven’t put all the function together in E. coli cells and coculture it with Propionibacterium acnes, we have constructed a model to simulate this situation. The growth simulator model predicts the trend of the change of our coculture system and gives feedback to how to modify our product, considering it as a whole. Through this model, we conclude that our design is quite effective and we can improve the effect of our ultimate product by improving the stability of bacteriocins in the outside environment and improving the ability of our engineered bacteria to decompose fatty acids. The latter one is what we are doing in the project and the former one will be taken into consideration in the future lab work.
