Difference between revisions of "Part:BBa K1884012"
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<partinfo>BBa_K1884012 short</partinfo>
 
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We provided Hygromycin B resistance under the RBCS2 promoter(BBa_K1884010)
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We provided Hygromycin B resistance under the RBCS2 promoter([https://parts.igem.org/Part:BBa_K1884010 BBa_K1884010])
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===Biology===
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RBCS2 is a promoter of the small subunit of ribulose-1, 5-diphosphate carboxylase, encoded by the nuclear gene and is an inducible promoter for strong light-induced expression. 
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Hygromycin is active against both prokaryotic and eukaryotic cells. It acts by inhibiting polypeptide synthesis. It stabilizes the tRNA-ribosomal acceptor site, thereby inhibiting translocation. .
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RBCS2 terminator is Chlamydomonas reinhardtii RuBisCO small subunit 2 terminator which obtained from the pOpt_mVenus_Hyg expression plasmid provided by Chlamydomonas Resource Center.<b>(Fig 1)</b>
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<figure style="text-align: center"><img style="width:70%" src="https://static.igem.org/mediawiki/2016/1/12/RHRzhili.png"/><figcaption style="text-align:center"><b>Figure 1.</b> The diagram of this composite part in pSB1C3 Backbone. </figcaption></figure>
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===Usage===
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For testing the efficiency of Hygromycin B, we formulated six gradient concentrations of Hygromycin B solution and add 200ul each into six different beakerflask which contain 200ml LB solid medium, then we put the competent cell into six different LB plate. there is a photograph to show several monoclonal colony growing on a LB plate which has the best selected abilty of Hygromycin B.<b>(Fig 2)</b>
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<figure style="text-align: center"><img style="width:70%" src="https://static.igem.org/mediawiki/2016/e/e6/Dankelongjun1.jpg"/><figcaption style="text-align:center"><b>Figure 2.</b> The conlony of E. coli in LB medium plate containing Hygromycin B. </figcaption></figure>
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Conbined with another resistant device(BBa_K1884011), we are able to screen the green algae which has paromoycin resistance and Hygromycin B resistance. we added 200ul paromoycin solution and hygromycin B solution into 200ml TAP solid medium, then we put the green algae without cytoderm into TAP plate. there is a photograph to show several colony growing on a TAP plate which has the best selected efficiency of paromoycin and Hygromycin B.<b>(Fig 3)</b>
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<figure style="text-align: center"><img style="width:70%" src="https://static.igem.org/mediawiki/2016/d/d3/HYGpingban.png"/><figcaption style="text-align:center"><b>Figure 3.</b> The conlony of green algae selected by paromoycin and Hygromycin B. </figcaption></figure>
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===Usage and Biology===
 
  
  

Latest revision as of 00:22, 20 October 2016


RBCS2 Promoter+Hygromycin B+RBCS2 Terminator

We provided Hygromycin B resistance under the RBCS2 promoter(BBa_K1884010)

Biology

RBCS2 is a promoter of the small subunit of ribulose-1, 5-diphosphate carboxylase, encoded by the nuclear gene and is an inducible promoter for strong light-induced expression.

Hygromycin is active against both prokaryotic and eukaryotic cells. It acts by inhibiting polypeptide synthesis. It stabilizes the tRNA-ribosomal acceptor site, thereby inhibiting translocation. .

RBCS2 terminator is Chlamydomonas reinhardtii RuBisCO small subunit 2 terminator which obtained from the pOpt_mVenus_Hyg expression plasmid provided by Chlamydomonas Resource Center.(Fig 1)

Figure 1. The diagram of this composite part in pSB1C3 Backbone.

Usage

For testing the efficiency of Hygromycin B, we formulated six gradient concentrations of Hygromycin B solution and add 200ul each into six different beakerflask which contain 200ml LB solid medium, then we put the competent cell into six different LB plate. there is a photograph to show several monoclonal colony growing on a LB plate which has the best selected abilty of Hygromycin B.(Fig 2)

Figure 2. The conlony of E. coli in LB medium plate containing Hygromycin B.

Conbined with another resistant device(BBa_K1884011), we are able to screen the green algae which has paromoycin resistance and Hygromycin B resistance. we added 200ul paromoycin solution and hygromycin B solution into 200ml TAP solid medium, then we put the green algae without cytoderm into TAP plate. there is a photograph to show several colony growing on a TAP plate which has the best selected efficiency of paromoycin and Hygromycin B.(Fig 3)

Figure 3. The conlony of green algae selected by paromoycin and Hygromycin B.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 650
    Illegal NgoMIV site found at 832
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1105