The design of Acneraser encompasses four major aspects, Inhibition Module, Fatty Acid Sensing Module, Fatty Acid Consumption Module and Safety Module. With four aspects integrated together, we envision "Acneraser" will become a novel and efficient therapy for acne. The experimental workflows and achievements are mainly summarized in Engineering Success.

Fig 1. Plasmid design overview

Notably, to demonstrate the biological designs mostly away from the laboratories, our team harness interdisciplinary methods and real-world perspectives for developing "Acneraser", which are shown in relevant pages.

Inhibition Module

Propionibacterium acnes is the pathogen of acne. It degrades sebum into fatty acid and its antigen can cause local inflammation, resulting in papule and even pustule. To specifically inhibit Propionibacterium acnes without causing too much influence on skin flora, we find bacteriocin PctA as our P. acnes inhibitor. PctA specifically targets Propionibacterium bacteria, including Propionibacterium acnes. [1][2] It will not kill E. coli, which means that it will not prevent our engineered bacteria from realizing other functions. For the delivery of PctA, the bacteriocin precursor contains a signal peptide sequence, which can help the secretion of bacteriocin into the environment and directly interact with Propionibacterium acnes. [1] In later experiments, we changed the original signal peptide into OmpA signal peptide to attain a higher efficiency for secretion. [3]

In this part of the design, we expected to firstly use the expression system of T7 promoter to produce bacteriocin PctA (with His tag) and test its secretion and function. First, we constructed the plasmid vector in DH5α strain, and then transferred it to BL21 strain for induced expression. After affinity purification, we tested the growth inhibition effect of the purified PctA on P. acne by the measurement of OD600 and inhibition zone experiment. The secretion of PctA is verified by Western Blot of culture supernatant. In the final version of our project, the expression of PctA gene will be controlled by the fatty acid sensing module described next.

Fig 2. Plasmid design for producing bacteriocin PctA

Fatty Acid Sensing Module

It is worth noting that Propionibacterium acnes is a conditional pathogen, which is also one of the main components of skin flora. A robust interference may disturb the balance of the flora, and cause undesired side effects. [4] And sebum is a natural protective barrier of the skin, which plays an significant role as well. So in order to maintain a balanced, symbiotic relationship between the skin and skin microbiota, we have to take control of the over-expression of two β-oxidation related enzymes and the expression of bacteriocin PctA exactly in our skin (only when the concentration of fatty acids is over the threshold can the engineered bacteria produce the bacteriocin PctA to inhibit the growth of over-proliferated P. acnes). We thus designed this module and selected the fatty acid-sensitive promoter pFadD_Lac, in the hope of providing a relatively accurate control over the yield of bacteriocin. In the absence of fatty acids, an intrinsic transcription factor FadR acting as the repressor in the fatty acid metabolism system in E. coli. will attach to the promoter, and block the downstream gene expression. This has been proved to be effective by a previous iGEM team (NTHU_Taiwan). [5]

Fig 3. pFadD_Lac promoter

We used GFP to test the performance of the promotor by measuring the fluorescence intensity. We set a series of time and concentration gradient to find out how those 2 factors influence the promotor. Moreover, according to the fact that most RBS with higher binding efficiency had the repetitive sequence of "AGGG", or "AAA" after the start base, we improved a new part of RBS and tried to compare it with the original RBS (BBa_B0030) to see which is better in some specific occasions.

Fig 4. The system to test the performance of the Fatty Acid Sensing Module at the beginning

Additionally, to better reduce expression leakage, we constitutively overexpressed FadR, which has been shown effective to increase bacterial sensitivity to fatty acids. [5] So our complete design includes the pFadD_Lac promoter and the overexpression of FadR.

Fig 5. Plasmid design for testing the performance of the Fatty Acid Sensing Module after optimization

Moreover, we designed a directed evolution pipeline to further optimize the function of the repressor FadR. Directed evolution (DE) is a method used in protein engineering that harnesses the power of selection to evolve molecules, typically proteins or nucleic acids, from large, stochastically permuted pools or combinatorial libraries, to have desired properties. It mainly comprises the following steps:

(1) Gene diversification by random mutagenesis and/or gene recombination to generate a diverse library of variants;

(2) Screening/selection to obtain variants with improved phenotypes.

(3) The improved variant will serve as a new starting point for the next round of gene diversification. [6]

Fig 6. The steps of directed evolution

Here, we used Error-prone PCR (epPCR) to generate a diverse library of FadR variants and then transformed with the system which we had built above into the same engineered bacteria to test its function. Compared with the original type of FadR, we can obtain a system with better performance and decide FadR that can be used in different situations (in a serious or a mild situation).

Fatty Acid Consumption Module

Propionibacterium acnes inhabits in pores and sebaceous glands which are anaerobic environments created by sebum. Researches have proved the role of sebum as an important ecological factor for the growth of P. acnes [7] and free fatty acids and triglycerides account for the major portion of the sebum[8], so one of the major features of our engineered bacteria is decomposing and utilizing fatty acids efficiently. On the one hand, it significantly cleans up oil and sebum on the user's faces, reducing stimulation to the skin. On the other hand, the decomposition of sebum unblocks the pores, destroying the anaerobic environment on which P. acnes rely to survive.

Fig 7. Plasmid design for the over-expression of two β-oxidation relating enzymes

Our engineered bacteria's ability to decompose fatty acid is strengthened by the over-expression of two β-oxidation related enzymes: fatty acyl-CoA synthetase (FadD) and fatty acyl-CoA dehydrogenase (FadE). (from iGEM 2019 BNU-China). And we will use fatty acid-sensitive promoter pFadD_Lac to prevent the excessive decomposition of sebum. Although we use the same promoter in Fatty Acid Consumption Module and Inhibition Module, but we use different plasmids to control the expression of the two parts.

Fig 8. Fatty acid β-oxidation pathway[9]

Safety Module

Considering we are setting up a design that will be used in human medicine, the safety of our engineered bacteria should be stressed. Auxotroph is a mutant microorganism that requires some nutrient not normally required by the original organism (prototroph). [10] In terms of safety, we will prevent environmental pollution or other safety threats by using glycine deficient engineered bacteria. In the process of searching the literature, we found several common types of gene defects. We finally chose glyA, which encodes protein serine hydroxymethyltransferase, as the targeted gene after several rounds of DBTL cycles. For more details. In practice, glycine will be added to the matrix that carries engineered bacteria. No place outside the matrix will the engineered bacteria be able to survive.

Fig. 9 Safety procedure using λ-Red system

We conducted the knock-out of glyA in our chassis with the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. The Red system includes three genes: γ, β, and exo, producing the proteins Gam, Bet, and Exo, respectively. Gam inhibits the host RecBCD exonuclease V so that Bet and Exo can gain access to DNA ends to promote recombination. [11] In our experiments, pKD46 was used to express the λ-Red recombinase system and pKD13 was used as a template for cloning resistance genes.

Fig. 10 Plasmid design for gene knockout


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