Part:BBa_K4711026

T7+alkB+alkG+T7+alkT+Spycatcher

Usage and Biology

Colony PCR results showed the appearance of the target band at around 4000 bp, indicating successful plasmid integration into Escherichia coli. SDS-PAGE analysis revealed the successful expression of the AlkB, AlkG, and AlkT proteins. Additionally, by setting a gradient of IPTG concentrations, it was observed that the protein expression was most efficient when induced with 0.5 mM IPTG, yielding the highest protein expression levels.

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 (56KDa), alkB (45KDa), and alkG (18KDa).

Source

Pseudomonas oleovorans

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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1860
  Illegal NheI site found at 3534
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1634
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 167
  Illegal NgoMIV site found at 1079
  Illegal NgoMIV site found at 2554
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1966