Difference between revisions of "Part:BBa K4006013"

 
 
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<partinfo>BBa_K4006013 short</partinfo>
 
<partinfo>BBa_K4006013 short</partinfo>
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===Background===
  
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This composite part includes coding sequences for the proteins metallothionein, arsenate reductase, and the protein ferritin, all codon optimized for use in the ''Chlamydomonas reinhardtii'' chloroplast. They were also each tagged with a different epitope tag, for the eventual purpose of protein expression evaluation by Western blot. The intention was for them to work synergistically to improve the uptake of arsenic out of contaminated water.
  
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===Cloning into ''E. coli'' and Verification===
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===Usage and Biology===
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The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent ''E. coli'' cells from NEB. The “Tagged” sequence, MT/6xHIS-IEE2-ArsC/HA-IEE5-FtnA/FLAG, was designed by our team and produced in two parts by IDT. However, our team did not have any success cloning this construct via golden braid. This may be due to the presence of IEE2, which is also present in the pASapI backbone. We were unable to confirm cloning via restriction digest and often did not get significant results from the transformation into 10-beta competent ''E. coli'' cells.
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4006013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4006013 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
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<partinfo>BBa_K4006013 parameters</partinfo>
 
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Latest revision as of 00:32, 22 October 2021


MT-6XHis-IEE5-arsC-HA-IEE2-FLAG-fTNa

Background

This composite part includes coding sequences for the proteins metallothionein, arsenate reductase, and the protein ferritin, all codon optimized for use in the Chlamydomonas reinhardtii chloroplast. They were also each tagged with a different epitope tag, for the eventual purpose of protein expression evaluation by Western blot. The intention was for them to work synergistically to improve the uptake of arsenic out of contaminated water.

Cloning into E. coli and Verification

The constructs were designed by our team and produced by IDT. They were transformed into 10-Beta competent E. coli cells from NEB. The “Tagged” sequence, MT/6xHIS-IEE2-ArsC/HA-IEE5-FtnA/FLAG, was designed by our team and produced in two parts by IDT. However, our team did not have any success cloning this construct via golden braid. This may be due to the presence of IEE2, which is also present in the pASapI backbone. We were unable to confirm cloning via restriction digest and often did not get significant results from the transformation into 10-beta competent E. coli cells. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 589
    Illegal BamHI site found at 45
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 5
    Illegal SapI site found at 685
    Illegal SapI site found at 1750
    Illegal SapI.rc site found at 233
    Illegal SapI.rc site found at 1150
    Illegal SapI.rc site found at 2305