Difference between revisions of "Part:BBa K3268004"

Revision as of 15:54, 16 October 2019

Strong expression of sfGFP in E. coli

To make a composite module that can highly express any inserted protein-coding genes, we had designed and constructed this strongly expressed sfGFP in E. coli as a proof of our concept.

1. The sfGFP coding region was PCR amplified from BBa_K1321337 using high-fidelity enzyme KOD-Plus.
2. The strong promoter along with BBa_B0034 RBS and BBa_B0015 transcriptional terminator on pSB1C3 plasmid backbone were PCR amplified from BBa_J364007 using high-fidelity enzyme KOD-Plus.
3. In order to make the strong promoter (along with BBa_B0034 RBS and BBa_B0015 transcriptional terminator on pSB1C3 plasmid backbone) useful to highly express inserted protein-coding genes, we had designed two PCR primers with HindII and BamHI sites respectively to generate a composite expression module without the original GFP coding sequence.
4. Any inserted protein-coding genes not containing HindII and BamHI sites in their coding sequences can then be amplified and cloned into our composite expression module improved from BBa_J364007.