Part:BBa_K5175032
T7 promoter-fucO-aldA-T7 terminator
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 241
Illegal PstI site found at 1989
Illegal PstI site found at 2702 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2760
Illegal PstI site found at 241
Illegal PstI site found at 1989
Illegal PstI site found at 2702 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1527
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 241
Illegal PstI site found at 1989
Illegal PstI site found at 2702 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 241
Illegal PstI site found at 1989
Illegal PstI site found at 2702
Illegal AgeI site found at 957
Illegal AgeI site found at 1158
Illegal AgeI site found at 1799 - 1000COMPATIBLE WITH RFC[1000]
Description
It is a composite component consisting of the T7 promoter, lac operator, target genes fucO, aldA. It is responsible for enabling E.coli to increase E.coli 's ability to efficiently utilise EG. EG is converted to glycolic acid (GA) in engineered E.coli. GA can be metabolized by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of H. coli by condensing with succinate via isocitrate lyase (encoded by the aceA gene), forming isocitrate.
Usage and Biology
EG is first converted in E.coli to glycolaldehyde (GLA) by L-1,2 -propylene glycol oxidoreductase, which is subsequently converted to glycolic acid (GA) by aldehyde dehydrogenase A. GA can be metabolized by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of H. coli by condensing with succinate via isocitrate lyase (encoded by the aceA gene), forming isocitrate
fucO
fucO is the gene for L-1,2-propanediol oxidoreductase, which is an iron-dependent group III dehydrogenase and can convert Ethylene glycol(EG) to glycolaldehyde (GLA).
aldA
aldA is the gene for aldehyde dehydrogenase A, which is an enzyme with a relatively broad substrate specificity for small hydroxyaldehyde substrates and can convert glycolaldehyde (GLA) to glycolic acid (GA).
Molecular cloning
Initially, we transformed the company-synthesized plasmids containing designed sequences into E.coli DH5α for amplification, allowing us to obtain a sufficient quantity of plasmid DNA for subsequent experiments. Following this, colony PCR was performed to confirm successful transformation, and the required plasmids were subsequently extracted for further experimentation. Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study.
The bands of pEG（~3000 bp）from PCR are identical to the theoretical lengths of 2862bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.
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