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Part: BBa_K1321337

sfGFP in Freiburg format (RFC 25)

This BBa_K1321337 contains just a coding sequence with no promoter to be expressed in E. coli.

  1. We were searching for fluorescent protein-coding genes to be used in our project. This BBa_K1321337 iGEM part was listed in the well 16K of Plate 5 of the 2018 Spring iGEM Distribution Kit. The plasmid backbone listed was pSB1C3 (see http://parts.igem.org/partsdb/get_part.cgi?part=BBa_K1321337). We had tried to do transformation with the provided plasmid DNA on E. coli DH5_alpha cells and grow the transformed cells on Chloramphenicol plates. However, no sfGFP color had shown up on the plates with and without UV or LED irradiation. Therefore, we would like to let other iGEM teams know that this BBa_K1321337 contains just a coding sequence with no promoter to be expressed in E. coli.
  2. Therefore, we went on to make an improved composite part that can strongly express this sfGFP protein.
  3. That is our Part: BBa_K3268004.

>> See details on Parts Registry

Part: BBa_K3268004

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.

>> See details on Parts Registry

Fig 1. Plasmid map of BBa_K3268004.