Difference between revisions of "Part:BBa K4711027"

 
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=Usage and Biology=
 
=Usage and Biology=
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Colony PCR results showed the appearance of target bands at approximately 4000 bp and 1000 bp, indicating successful plasmid integration into Escherichia coli. SDS-PAGE analysis revealed successful expression of the AlkB, AlkG, AlkT, and GDH proteins. By setting a gradient of IPTG concentrations, it was observed that the protein expression was most efficient when induced with 0.2 mM IPTG, yielding the highest protein expression levels.
  
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===Source===
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Moreover, a distinct band was observed at around 90 KDa, indicating successful linkage of alkT and GDH through SpyTag and SpyCatcher. As a result, the band corresponding to GDH was lighter compared to the previous one due to partial migration to the 90 KDa position as a result of binding with GDH.
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  <figure>
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      <img src="https://static.igem.wiki/teams/4711/wiki/results/figure-11.png"width="100%" style="float:center">
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      <figcaption>
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Fig 1 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG 3: 0.5mM IPTG 4: 1mM IPTG.The protein bands from top to bottom are alkT+GDH (87KDa), alkT (56KDa), alkB (45KDa), GDH (31KDa), and alkG (18KDa).
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      </figcaption>
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  </figure>
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===Source===
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Pseudomonas oleovorans
 
===Potential applications===
 
===Potential applications===
  
 
===References===
 
===References===
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[1]Qiaofei He, George N. Bennett, Ka-Yiu San, Hui Wu*. Biosynthesis of medium-chain ω-hydroxy fatty acids by AlkBGT of Pseudomonas putida GPo1 with native FadL in engineered Escherichia coli. Frontiers in Bioengineering and Biotechnology. 2019. 7:273.
  
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[2] Staijen I E , Beilen J B V , Witholt B .Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli[J].European journal of biochemistry, 2000, 267(7):1957-65.DOI:10.1046/j.1432-1327.2000.01196.x.
 
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===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 09:11, 10 October 2023


T7+GDH+SpyTag

Usage and Biology

Colony PCR results showed the appearance of target bands at approximately 4000 bp and 1000 bp, indicating successful plasmid integration into Escherichia coli. SDS-PAGE analysis revealed successful expression of the AlkB, AlkG, AlkT, and GDH proteins. By setting a gradient of IPTG concentrations, it was observed that the protein expression was most efficient when induced with 0.2 mM IPTG, yielding the highest protein expression levels.

Moreover, a distinct band was observed at around 90 KDa, indicating successful linkage of alkT and GDH through SpyTag and SpyCatcher. As a result, the band corresponding to GDH was lighter compared to the previous one due to partial migration to the 90 KDa position as a result of binding with GDH.

Fig 1 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG 3: 0.5mM IPTG 4: 1mM IPTG.The protein bands from top to bottom are alkT+GDH (87KDa), alkT (56KDa), alkB (45KDa), GDH (31KDa), and alkG (18KDa).

Source

Pseudomonas oleovorans

Potential applications

References

[1]Qiaofei He, George N. Bennett, Ka-Yiu San, Hui Wu*. Biosynthesis of medium-chain ω-hydroxy fatty acids by AlkBGT of Pseudomonas putida GPo1 with native FadL in engineered Escherichia coli. Frontiers in Bioengineering and Biotechnology. 2019. 7:273.

[2] Staijen I E , Beilen J B V , Witholt B .Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli[J].European journal of biochemistry, 2000, 267(7):1957-65.DOI:10.1046/j.1432-1327.2000.01196.x. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]