Difference between revisions of "Part:BBa K4011003:Design"

 
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
We use a rigid linker with the protein sequence EAAAKEAAAK to fuse fre and sttH. SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp into 6-X-trp, and is highly insoluble in E. coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E. coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added.
+
1. All codons were optimized for <i>E. coli</i> based on <i>E. coli</i> codon bias.
−
 
+
−
To express Fre-SttH, we first attempted the T7 system, commonly used in E. coli BL21(DE3). We expressed histag-Fre-SttH using the T7 system with a lacO promoter, but found both its expression and solubility to be fairly low, thus making it unsuitable for our project.
+
−
 
+
−
Because we needed a knockout TnaA (TnaA is naturally expressed in E. coli, giving it its characteristic scent) strain to express Fre-SttH so that our natural trp will not be converted to indole without being halogenized by Fre-SttH, we switched from the T7 system to the ptac system. The ptac system, unlike the T7 system which can only be used in E. coli BL21, can be used in all E. coli strains. Our knockout strain was supplied in E. coli DH5&#945;, courtesy of Sha Zhou, in which we constructed ptac-histag-Fre-SttH and ptac-Fre-SttH.
+
  
 +
2. We used a rigid linker with the protein sequence EAAAKEAAAK to fuse Fre and SttH.
  
 +
3. We first added his-tag to Fre-SttH, however only to find that its expression is very low and insoluble in both T7 and ptac systems.
  
 +
4. We first attempted the T7 system, which is commonly used in <i>E. coli BL21(DE3)</i> to express Fre-SttH. However we found that its expression and solubility to be fairly low, we then switched to the ptac system which can be used in all <i>E. coli</i> strains.
  
 +
5. The ptac system allows us to express Fre-SttH in a knockout TnaA strain <i>DH5α</i>(TnaA is naturally expressed in <i>E. coli</i>, giving it its characteristic scent) so that our natural trp will not be converted to indole without being halogenized by Fre-SttH.
  
  
 
===Source===
 
===Source===
  
−
Fre-SttH is composed of two separate domains, fre and sttH.  Fre is from E.coli and SttH is from Streptomyces toxytricini.
+
Fre-SttH is composed of two separate domains: fre and sttH.  Fre is from <i>E.coli</i> and SttH is from <i>Streptomyces toxytricini</i>.
  
 
===References===
 
===References===

Revision as of 03:43, 21 October 2021


Fre-SttH


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 576
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 948
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 424


Design Notes

1. All codons were optimized for E. coli based on E. coli codon bias.

2. We used a rigid linker with the protein sequence EAAAKEAAAK to fuse Fre and SttH.

3. We first added his-tag to Fre-SttH, however only to find that its expression is very low and insoluble in both T7 and ptac systems.

4. We first attempted the T7 system, which is commonly used in E. coli BL21(DE3) to express Fre-SttH. However we found that its expression and solubility to be fairly low, we then switched to the ptac system which can be used in all E. coli strains.

5. The ptac system allows us to express Fre-SttH in a knockout TnaA strain DH5α(TnaA is naturally expressed in E. coli, giving it its characteristic scent) so that our natural trp will not be converted to indole without being halogenized by Fre-SttH.


Source

Fre-SttH is composed of two separate domains: fre and sttH. Fre is from E.coli and SttH is from Streptomyces toxytricini.

References