Overview
This year Team KEYSTONE aims to produce create a bio-body deodorant with sandalwood aroma which is mainly composed of fengycins and santalene. In our project, our modification on Bacillus subtilis 168 could provide reference for gene editing on Bacillus subtilis and biosynthesis of lipopeptides. Our measurement of fengycins production by HPLC could be utilized for quantitative determination of fengycins. Furthermore, fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus hemolyticus is verified, which indicates the potential of Fengycins as a novel antibiotic. In the aspect of sandalene synthesis, we proposed 4 new composite parts, and also successfully validating our improvement on the existing part ERG20 (Part number: BBa_K849001). Last but not least, we have designed a domestic deodorizing fermentation tank, enriching the application scenario of our products.
Modification of Bacillus subtilis 168 produce fengycins
With the help of pJOE8999_sfp_degQ plasmid (Figure 1a), we knocked in both sfp gene and degQ gene in the region of the natural sfp gene of Bacillus subtilis 168 after being knocking out. Using composite part BBa_K4274035, we successfully modified the genome of Bacillus subtilis 168, which is conveyed by the electrophoresis result (Figure 1b) and the sequencing result (Figure 2a). And the HPLC result have illustrated that our modification of Bacillus subtilis 168 enables the strain to produce fengycins.
Visit our Results Page for more information.
Figure 1. Edting the genome of Bacillus subtilis 168 to enable it to produce fengycins. (a) Plasmid design for knocking out the invalid sfp gene of Bacillus subtilis 168 and knocking in the sfp gene from B. amyloliquefaciens FZB42 and degQ gene from Bacillus subtilis 168. (b) Electrophoresis results show that gene editing is successful. (c) Protocol about transformation, induction and elimination of pJOE8999 plasmid in Bacillus subtilis 168.
Measurement of fengycins production by HPLC
For the quantification of fengycins production, various concentrations of fengycins standards were analyzed by HPLC. According to their peak area, the standard curve and its corresponding regression equation y=9.767x+193.44 is obtained, and R2>0.98 displays that the regression equation fits the observed values (Figure 2b). Using our detection method of HPLC, the regression equation can be utilized for quantitative determination of fengycins.
Visit our Experiments Page for more information about protocols of HPLC analysis.
Figure 2. Quantification analysis of fengycins production. (a) The sequencing result of engineering Bacillus subtilis 168 strain for production. (b) The standard curve of peak area obtained by HPLC (y) and its corresponding fengycins’ concentration (x). (c) HPLC results of sample and various concentrations of fengycins standard.
Verification of fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus hemolyticus
Based on the experiments of biofilm quantitative detection and growth curve measurement, we verified that 1.25 g/L of lipopeptide extract (containing 5.625μM fengycins) would be the optimal inhibitory concentration to be applied in our product, which could result in a reduction of about 20% in biofilm content of S. haemolyticus, and also no inhibitory effect on the growth of S. hemolyticus. It also indicates the potential of Fengycins as a novel antibiotic in the pharmaceutical industry.
Figure 3. Fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus haemolyticus. (a) Biofilm quantitative detection of Staphylococcus hemolyticus at different concentrations of lipopeptide extract. (b) Measurement of the growth curve of Staphylococcus hemolyticus at different concentrations of lipopeptide extract.
Improvement of ERG20 related to santalene synthesis
With the help of the co-transformation of pMVA plasmid with various pW1 plasmids, including pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL, different strains were successfully constructed and utilized for santalene production. The yield of santalene from strains CE, CEM, TCEM, CEM_FL could be used to characterize composite parts BBa_K4274030, BBa_K4274031, BBa_K4274032, BBa_K4274033. Eventually, strain CEM produces the maximal level of α-santalene, which elucidates that the mutation of 96th amino acid into tryptophan could increase the yield of α-santalene by about 20%. It substantiated the prominent performance of ERG20_F96W in enhancing the supply of FPP and α-santalene production in E. coli.Visit our Results Page for more information.
Figure 4. Characterization of BBa_K4274030, BBa_K4274031, BBa_K4274032, BBa_K4274033. (a) Measurement santalene production of different strains by GC/MS results. (b) Schematic representing the structure of pMVA, pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL transformed into E.coli DH5α ∆TnaA.
Domestic Deodorant Fermenter
We designed a small-size bioreactor that provides a constant supply of a solution of antibacterial deodorant, fengycin, which can be applied to the human body through a spray bottle or a water-absorbing patch. The special “bridge” design of our hardware allows us to achieve two-media culturing with a minimal use of complex industrial design and control units, facilitating a stable performance in any household environment. Furthermore, we also invented a unique way of confining bacteria during the functioning of the product that maximizes its bio-safety, efficiency of production, and suitability for compact packaging and longtime storage. In addition, mathematic modeling was incorporated into the development of hardware as it provides necessary parameters like how long show the bacteria stay in on medium and estimates the overall efficiency of fengycin production. This product is durable, easy-to-maintain, and easy-to-use, which fit precisely to its purpose: our hardware will become a “home appliance” as useful as the others that brings synthetic biology into people’s daily lives and improves their lives with it.
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Figure 5. The Schematic representaion of Domestic Deodorant Fermenter