Results are derived after 5 times of Gibson Assembly, over 150 miniprep plasmid purification, nearly 100 gel electrophoresis and over 150 plates spreaded..
Using The Inoue Method for Preparation and Transformation of Competent E. coli, all constructions are transformed into E. coli BL21 and Neb 5 alpha. Plasmid purification are done using the TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 and Monarch® Plasmid Miniprep Kit.pETBlue-2 is used for Blue-White screening.
With reference to Fig. A below, it is observed that in the 3rd lane after an hour of 42V gel electrophoresis, a comparably larger fragment is constructed using Gibson Assembly.
All constructs in Fig. B are comparably larger products assembled by Gibson Assembly. The circular DNA on the 5th lane to the right is plasmid purified Gibson assembly product and the 4th lane from the right is its linearized form using double digestion EcoRI and PstI. This indicates that the product is Bio-Brick compatible. In addition, after several hours of gel electrophoresis with 42V, except for these two lanes mentioned above, all other bands migrate downwards to the 8kbp point after one hour. This concludes that among all constructs we have done, this product matches the most with our expectation.
These shows that we have successfully assembled the nif cluster using the Gibson Assembly.
As the high GC content make it difficult to complete PCR, we amplified the PETase and MHETase with PCR after codon optimization. For the reason that the band of MHETase is weak, we then try to perform gradient PCR using 6 different temperatures. After ligation and transformation, only PETase is successfully transformed. We express the protein and measured the size of it. With the help of HKUST team, the PETase is characterized using SDS-Page.