Difference between revisions of "Part:BBa K4244003"
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<partinfo>BBa_K4244003 short</partinfo>
 
<partinfo>BBa_K4244003 short</partinfo>
  
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sfGFP <partinfo>BBa_K4244004</partinfo> was fused to PelB <partinfo>BBa_J32015</partinfo> (fused at the N-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].
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====Secretion assay====
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[[File:BBa_K4244003-PelB-sfGFP.png|thumb|500px|center|<font size="1"> Figure 1: End point measurement of total, pellet and supernatant fraction of sfGFP under the PelB 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 ''Escherichia coli'' Nissle 1917. This experiment still need adjustment and fine tuning to prove the secretion of sfGFP.
  
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[[File:BBa_K4244003-PelB-sfGFP.png|200px|center]]
 
  
 
Endpoint measurement  
 
Endpoint measurement  
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<partinfo>BBa_K4244003 parameters</partinfo>
 
<partinfo>BBa_K4244003 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.

Latest revision as of 12:55, 12 October 2022


Rhamnose indicuble PelB:sfGFP

sfGFP BBa_K4244004 was fused to PelB BBa_J32015 (fused at the N-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 PelB 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 need adjustment and fine tuning to prove the secretion of sfGFP.


Endpoint measurement

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 193
  • 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.