Difference between revisions of "Part:BBa K4244013"

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<partinfo>BBa_K4244013 short</partinfo>
 
<partinfo>BBa_K4244013 short</partinfo>
  
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sfGFP <partinfo>BBa_K4244004</partinfo> was fused to HlyA <partinfo>BBa_K554002</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 [1].  
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sfGFP <partinfo>BBa_K4244004</partinfo> was fused to HlyA <partinfo>BBa_K554002</partinfo> (fused at the C-terminus) 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 [1].  
  
 
====Secretion assay====
 
====Secretion assay====
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====Discussion====
 
====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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No secretion was observed in ''Escherichia coli'' Nissle 1917. This experiment still needs adjustment and fine tuning to prove the secretion of sfGFP.
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Latest revision as of 12:47, 12 October 2022


Rhamose inducible sfGFP with HlyA secretion tag

sfGFP BBa_K4244004 was fused to HlyA BBa_K554002 (fused at the C-terminus) 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 [1].

Secretion assay

Figure 1: End point measurement of total, pellet and supernatant fraction of sfGFP under the HlyA 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 Escherichia coli Nissle 1917. This experiment still needs 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.