Composite

Part:BBa_K4711026

Designed by: Qingyu Wu   Group: iGEM23_OUC-China   (2023-09-22)
Revision as of 03:41, 11 October 2023 by Wuqingyu (Talk | contribs) (→‎Usage and Biology)

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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.

During the induction of expression for these three proteins, we observed significant differences in their expression levels. However, the RBS we used is already strong for these three proteins. Despite this, it still caused significant variations in protein expression, which can have an impact on the final fermentation catalysis. Due to time constraints, we were unable to redesign the RBS for further experimentation. Therefore, we suggest that future teams intending to use this part should redesign the RBS to ensure minimal differences in expression levels for these three proteins.

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

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
    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


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