Difference between revisions of "Part:BBa K5175003"
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<h1>'''Usage and Biology'''</h1> | <h1>'''Usage and Biology'''</h1> | ||
| − | The final products of PET degradation by the two-enzyme system are TPA and EG. However, wild-type E.coli cannot rapidly utilise these substances for various life activities.In order to increase the efficiency of E. coli in utilising the PET degradation products and to improve its viability, we overexpressed L-1,2-propanediol oxidoreductase and aldehyde dehydrogenase A.This modification was able to increase E.coli 's ability to efficiently utilise EG. | + | The final products of PET degradation by the two-enzyme system are TPA and EG. However, wild-type <i>E.coli</i> cannot rapidly utilise these substances for various life activities.In order to increase the efficiency of <i>E.coli</i> in utilising the PET degradation products and to improve its viability, we overexpressed L-1,2-propanediol oxidoreductase and aldehyde dehydrogenase A.This modification was able to increase <i>E.coli</i>'s ability to efficiently utilise EG. |
| − | We chose fucO as the gene for L-1,2-propanediol oxidoreductase and aldA as the gene for aldehyde dehydrogenase A. L-1,2-propanediol oxidoreductase is an iron-dependent group III dehydrogenase, and aldehyde dehydrogenase A is an enzyme with a relatively broad substrate specificity for small hydroxyaldehyde substrates. 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 metabolised by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of | + | We chose fucO as the gene for L-1,2-propanediol oxidoreductase and aldA as the gene for aldehyde dehydrogenase A. L-1,2-propanediol oxidoreductase is an iron-dependent group III dehydrogenase, and aldehyde dehydrogenase A is an enzyme with a relatively broad substrate specificity for small hydroxyaldehyde substrates. EG is first converted in <i>E.coli</i> 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 metabolised by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of <i>E.coli</i> by condensing with succinate via isocitrate lyase (encoded by the aceA gene) , forming isocitrate. |
<h1>'''Molecular cloning'''</h1> | <h1>'''Molecular cloning'''</h1> | ||
| − | 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. | + | Initially, we transformed the company-synthesized plasmids containing designed sequences into <i>E.coli</i> 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. | Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study. | ||
| + | <html> | ||
| + | |||
| + | <figure><center> | ||
| + | <img | ||
| + | alt="" | ||
| + | src="https://static.igem.wiki/teams/5175/resources/result/result-01.png" | ||
| + | width="700" | ||
| + | title=""> | ||
| + | <figcaption>Fig 1.The bands of pPeteg-P (upper band) and pPeteg-M (lower band)（~3000 bp）from PCR<br> | ||
| + | <br>The bands of pPeteg-P (upper band) and pPeteg-M (lower band)（~3000 bp）from PCR are identical to the theoretical lengths of 2862 bp estimated by the designed primer locations (homologous recombination fragments), which could demonstrate that these plasmids had successfully been obtained.</figcaption> | ||
| + | </figure> | ||
| + | <html> | ||
| + | |||
| + | <figure><center> | ||
| + | <img | ||
| + | alt="" | ||
| + | src="https://static.igem.wiki/teams/5175/resources/result/result-03.png" | ||
| + | width="700" | ||
| + | title=""> | ||
| + | <figcaption>Fig 3.The bands of pEG（~3000 bp）from PCR<br> | ||
| + | <br>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.</figcaption> | ||
| + | </figure> | ||
Revision as of 22:36, 1 October 2024
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).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 167
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 167
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 167
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 167
Illegal AgeI site found at 883
Illegal AgeI site found at 1084 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The final products of PET degradation by the two-enzyme system are TPA and EG. However, wild-type E.coli cannot rapidly utilise these substances for various life activities.In order to increase the efficiency of E.coli in utilising the PET degradation products and to improve its viability, we overexpressed L-1,2-propanediol oxidoreductase and aldehyde dehydrogenase A.This modification was able to increase E.coli's ability to efficiently utilise EG. We chose fucO as the gene for L-1,2-propanediol oxidoreductase and aldA as the gene for aldehyde dehydrogenase A. L-1,2-propanediol oxidoreductase is an iron-dependent group III dehydrogenase, and aldehyde dehydrogenase A is an enzyme with a relatively broad substrate specificity for small hydroxyaldehyde substrates. 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 metabolised by condensation with acetyl coenzyme A via the glyoxalate shunt to form malic acid. GA can also enter the metabolic pathway of E.coli by condensing with succinate via isocitrate lyase (encoded by the aceA gene) , forming isocitrate.
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 pPeteg-P (upper band) and pPeteg-M (lower band)（~3000 bp）from PCR are identical to the theoretical lengths of 2862 bp estimated by the designed primer locations (homologous recombination fragments), which could demonstrate that these plasmids had successfully been obtained.
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.