Difference between revisions of "Part:BBa K3268004"

 
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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.
 
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.
  
 
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<!-- Add more about the biology of this part here

Revision as of 11:25, 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 726
    Illegal NheI site found at 749
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1424
    Illegal SapI.rc site found at 10