Part:BBa_K3715023

T7 promoter+RBS+ His+Linker g+MBP+Linker c+PETase_MT9+Linker d+T7 terminator

This part consists of T7 promoter, RBS, protein coding sequence(His+Linker g+MBP+Linker c+PETase_MT9+ Linker d) and T7 terminator,and the biological module can be build into E.coli for protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 70
  Illegal PstI site found at 2080
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 70
  Illegal PstI site found at 2080
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 441
  Illegal XhoI site found at 2087
  Illegal XhoI site found at 2101
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 70
  Illegal PstI site found at 2080
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 70
  Illegal PstI site found at 2080
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 139
  

Usage and Biology

This composite part is made up with five basic parts, the RBS, two cutting sites NcoI and XhoI (linker c,d) , the His tag,and our target protein PETase_MT9. It encodes a protein which is PETase_MT9 fused with His-MBP tag. The fusion protein is about 31.49kD. In order to gain the highly purified target protein, we add His tag and MBP tag in N-terminal of PETase_MT9 and combine the two parts with the cutting site of protease. The fusion protein can be cut off at the cutting site by protease. It is convenient for us to purify our target protein.

Origin(organism)

Ideonella sakaiensis

Molecular cloning

First, we used the vector pMAT9s to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(50μg/mL) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 900ml LB medium and added antibiotic to 50 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 5-6 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-MBP tag fused with target protein.

• First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  
• Second, add the protein solution to the column, let it flow naturally and bind to the column.
  
• Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  
• Forth,add Ni-Washing buffer several times. Check as above. Collect the eluted proteins for further operation.
  

Figure 2. The result of SDS-PAGE.

Gel filtration chromatography:
The collected protein samples are concentrated in a 30 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 75 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

Figure 3. The result of gel filtration used the superdex75 column with the AKTA system.

Enzyme activity determination

We use HPLC equipment to measure the peak area of the product of PET (MHET) of the reaction, in order to express the enzyme activity of PETase. For more information on the product of PET(MHET), please see our project introduction.

Figure 4. Enzyme activity determination, compared with wild type.

Conclusion

Obviously, the enzyme activity of PETase_MT9 has significantly improved, compared with wild type.