Part:BBa_K4711027

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

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]