Difference between revisions of "Part:BBa K3268005"

 
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<partinfo>BBa_K3268005 short</partinfo>
 
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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 eforRed 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 eforRed in E. coli as a proof of our concept.
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1. The mCherry coding region was PCR amplified from BBa_J176005 using high-fidelity enzyme KOD-Plus.
 
1. The mCherry coding region was PCR amplified from BBa_J176005 using high-fidelity enzyme KOD-Plus.
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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.
 
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.
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<br>
 
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.
 
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.
 +
<br>
 
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.
 
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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<img src="https://static.igem.org/mediawiki/parts/f/fe/T--NYMU-Taipei_J364007_with_mCherry_CDS.png" width="60%" height="60%">
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 16:23, 20 October 2019


Strong expression of mCherry 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 eforRed in E. coli as a proof of our concept.
1. The mCherry coding region was PCR amplified from BBa_J176005 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 720
    Illegal NheI site found at 743
  • 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 1418