Difference between revisions of "Part:BBa K4244012"

 
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<partinfo>BBa_K4244012 short</partinfo>
 
<partinfo>BBa_K4244012 short</partinfo>
  
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sfGFP <partinfo>BBa_K4244004</partinfo> was fused to SEC-N22 <partinfo>BBa_K2895007</partinfo> to secrete sfGFP and use it as an extracellular reporter. It is under an inducible promoter <partinfo>BBa_K914003</partinfo>. For better titration using <partinfo>BBa_K914003</partinfo> we introduced the plasmid in a &Delta; ''rhaB'',&Delta; ''rhaT'' strain for better titration [1].
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====Secretion assay====
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[[File:BBa_K4244012-SEC-N22-sfGFP.png|thumb|500px|center|<font size="1">Figure 1: End point measurement of total, pellet and supernatant fraction of sfGFP under the SEC-N22 signal peptide expressed with rhamnose inducible promoter. Measurement was performed 5 hours after rhamnose induction at 500 μM. No significant fluorescence was detected in the supernatant fraction.</font>]]
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====Discussion====
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No secretion was observed in ''E. coli'' Nissle. This experiment still need adjustment and fine tuning to prove the secretion of sfGFP.
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<partinfo>BBa_K4244012 parameters</partinfo>
 
<partinfo>BBa_K4244012 parameters</partinfo>
 
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===References===
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1. Hjelm A, Karyolaimos A, Zhang Z, Rujas E, Vikström D, Slotboom DJ, et al. Tailoring Escherichia coli for the l -Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins. ACS Synth Biol. 2017;6(6):985–94.

Revision as of 08:20, 10 October 2022


Rhamose inducible sfGFP with SEC-N22 secretion tag

sfGFP BBa_K4244004 was fused to SEC-N22 BBa_K2895007 to secrete sfGFP and use it as an extracellular reporter. It is under an inducible promoter BBa_K914003. For better titration using BBa_K914003 we introduced the plasmid in a Δ rhaB,Δ rhaT strain for better titration [1].

Secretion assay

Figure 1: End point measurement of total, pellet and supernatant fraction of sfGFP under the SEC-N22 signal peptide expressed with rhamnose inducible promoter. Measurement was performed 5 hours after rhamnose induction at 500 μM. No significant fluorescence was detected in the supernatant fraction.

Discussion

No secretion was observed in E. coli Nissle. This experiment still need adjustment and fine tuning to prove the secretion of sfGFP.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Hjelm A, Karyolaimos A, Zhang Z, Rujas E, Vikström D, Slotboom DJ, et al. Tailoring Escherichia coli for the l -Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins. ACS Synth Biol. 2017;6(6):985–94.