Difference between revisions of "Part:BBa K3268004"
 
(3 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3268004 short</partinfo>
 
<partinfo>BBa_K3268004 short</partinfo>
  
 +
<br>
 
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.
 
1. The sfGFP coding region was PCR amplified from BBa_K1321337 using high-fidelity enzyme KOD-Plus.
 +
<br>
 
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.
 +
<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.
−
 
+
<br>
 +
<br>
 +
<html>
 +
<div>
 +
<img src="https://static.igem.org/mediawiki/parts/b/b5/T--NYMU-Taipei_pSB1C3_J364007_with_sfGFP_CDS%2Cjpg.jpeg" width="60%" height="60%">
 +
</div>
 +
</html>
 +
<br>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 07:06, 21 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