Team:HKJS S/Collaborations



After our discussion with the HKUST team, we attempt to optimize the Gibson Assembly used to inserted OmpA gene into the desired plasmid.

 Our design to attempt restriction enzyme digestion before we insert the OmpA gene into the plasmids. This can lower the difficulty for PCR amplification as some suggested that if the primers are too close to each other during a circular DNA amplification, it increases the difficulty. As Suggested, routine linearization of plasmid DNA increases the efficiency of PCR amplification as PCR amplification works optimal with relaxed DNA. Hence, we choose to linearize the plasmids before the Laccase and after the Ribosome Binding site using BseRI. Learning from the Nitrogenase Gibson Assembly in our project, which we constructed overlapping region on all fragments, we choose to construct all overlapping regions before Gibson Assembly in this design. This ensure the assembly is specific and prevent frameshift.


On the other hand, HKUST team helped us with the SDS-PAGE, characterizing our PETase. As the HKUST team is more experienced with protein extraction, we offered them PETase constructs, which are inserted to pETBlue-2 and pSB1C3, and the result returned is positive, showing that the NEB 5 Alpha strain managed to express the protein in both pETBlue-2 and pSB1C3.

The Collaboration between HKUST team and our team have improved the quality of both teams’ project.


Gibson Assembly

1.    Digest Plasmid with Bseri




1.     Set up reaction as follows:




1 µg

10X CutSmart Buffer

5 µl (1X)


1.0 µl (or 10 units)

Nuclease-free Water

to 50 µl

2.     Incubate at 37°C for 5–15 minutes as BseRI is Time-Saver qualified.

Please refer to!/protocol/re/single/BseRI

2.    PCR to remove the excess basepair on the plasmid

To ensure that all protein coding sequence is in frame to the T7 promoter.




pSB1c3_ Rev


 Annealing temp :68

Add 5μL of Q5® High GC Enhancer

3.    PCR to add overlapping complementary basepair ON OMPA sequence

 base pair complementary to the plasmid is added to both the beginning and the end of the OMPA sequence for subsequent Gibson Assembly.





Annealing temp :71

Add 5μL of Q5® High GC Enhancer

4.    Perform gibson assembly to insert OMPA

Standard Gibson Assembly


Please refer to Gibson Assembly from