Part:BBa_K3478890
bLIS((3R)-linalool synthase)
Summary
bLIS is a gene that expresses linalool synthase The linalool synthase used in our experiments is the linalool synthase from Streptromyces clavuligerus. linalool synthase is a enzyme that can effectively synthesize linalool
Description
bLIS is a gene that expresses linalool synthase The linalool synthase used in our experiments is the linalool synthase from Streptromyces clavuligerus. linalool synthase is an enzyme that can effectively synthesize linalool. we used Gibson assembly to combine LIS fragment and its vector pR6K, and transformed it in to E. Coli DH5α, and linalool synthase has successfully been produce. We used pTac promoter and lacI repressor in the plasmid, which means that IPTG can be used to induce the production of linalool synthase.
Figure 1: protein electrophoresis results of linalool synthase (65.6 kDa)
However, coding LIS need to work cooperate with GPPS and MVA which produces the substrate, GPP, for LIS. We used Gibson assembly to produce plasmid pR6k-ptac-GPPS-LIS and p15A and MVA. we transformed both plasmids into E.coli DH5α and conducted a colony polymerase chain reaction of E.coli transformed with pR6K-ptac-GPPS-LIS, which allows us to determine the success of Gibson assembly (figure 7A). Therefore, we successfully constructed and transformed pR6K-ptac-GPPS-LIS to E.coli DH5α.
Figure 2: results of Gibson assembly. (A) gel electrophoresis result of pR6K-ptac-GPPS-LIS (14358 bp). (B) gene sequencing result of p15A-MVA.
We cotransformed P15A-MVA and pR6K-ptac-GPPS-LIS plasmid to E.coli DH5α. E.coliDH5αwe induced them with IPTG and two of them with extra glucose. after the inducing process, the bacteria solution was treated with n-hexane to extract purified linalool(figure 9). We can clearly smell the strong fragrance of linalool in those samples, and samples with glucose have a stronger fragrance than samples that are not treated with glucose. This indicates that glucose may promote the synthesis of linalool in E.coli DH5α. We do not need to knock out the gene in E.coli producing stinky smell because the aroma overcomes the smell of E.coli. Therefore, our E.coli won’t affect the local environment and appearance.
Besides, we also compared the smell of our sample to standard linalool sample, and standard geraniol, which is the product of pR6K-ptac-GPPS-GES (the plasmid before our improvement) Based on our observation the aroma of geraniol contains sweetness; whereas, the aroma of standard linalool sample has a slight peppery smell. The two smells are quite distinguishable. The small of our samples also have a slightly peppery aroma, which is close to the linalool standard sample. Thus, we have successfully produced linalool.
In addition, through visual observation, we also confirmed that glucose can promote the synthesis of linalool in E.coli. The transparent liquids in the test tubes are purified linalool (figure 9). The volume of the liquid in samples treated with glucose is larger than samples that aren’t treated by glucose. Therefore, E.coli treated with glucose may be able to produce more linalool in the same condition than E.coli that is not. Our next step may be finding the optimal condition for linalool synthesis in E.coli. However, due to the Covid-19 situation, we do not have enough time in the lab, so we can only do those experiments in the future.
Figure 3: E.coli DH5α with P15A-MVA and pR6K-ptac-GPPS-LIS was propagated in four separated conical flasks. After the bacteria solutions reach the suitable OD (0.6-0.8), we added 2.5 ml glucose to two of the mediums during and added 25mM of IPTG to By adding n-hexane (4.5ml) to the induced bacteria solution, we can extract pure linalool (transparent layer)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 602
Illegal AgeI site found at 181
Illegal AgeI site found at 857 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 540
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