Part:BBa_K3868100

pET-MME-eGFP

pET--MME-eGFP plasmid contains pT7, lacO, MME, thrombin, eGFP-His-tag etc.Based on the plasmid of pET-24a, the C-terminal of MME was fused with eGFP in order to characterize the yield of MME. Moreover, in order to purify the MME , the enzyme loci of thrombin was inserted between MME and eGFP, and the label of 6*His was inserted at the end of eGFP, yielding the plasmid of pET-MME-eGFP. The fluorescence intensity of eGFP could be used to indicate the expression level of MME.

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]

Usage and Biology

        In this part, we still takes the dual plasmid expression system for genome editing. The sgRNA on the pTarget+Donor was designed to target the gene of CM (Fig.7A). The pCas and pTarget+Donor plasmids were co-transformed into BL21 (DE3)-CM via electroporation, then the single colonies were cultivated in LB medium with CM and non-CM, respectively. The transformants that cannot grow under CM condition were considered correct ant used for further sequencing (Fig.7B).

Results

         During construction of the library, 90 single colonies were randomly selected for sequencing. It was found that 48 variants with different RBS sequence were identified from 90 samples, with an editing efficiency of only 53%. However, it is noteworthy that the transformants were grown on the solid medium for longer time, the reproducibility of the results gradually increased, and majority of variant RBS sequences became GAAAAAAG (Fig.4), probably due to the continuous base editing in the transformants. The above results show that although CBE possesses the advantages of simplicity and rapidity, editing results and efficiency applied in BL21 (DE3) are not sufficiently stable.

Fig 7. A. The schematic diagram of pTarget+Donor and the composite part of BBa_K3868099. B. The whole process of constructing the library of BL21 (DE3)-derived variant strains using CRISPR/Cas9. C. The partial sequences and the G and A abundance variation maps of RBS variants obtained by CRISPR from three batches respectively.

Reference

Schlegel S, Löfblom J, Lee C, Hjelm A, Klepsch M, Strous M, Drew D, Slotboom DJ, De Gier JW, Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21 (DE3). Journal of molecular biology. 2012; 423: 648–659.