Difference between revisions of "Part:BBa K1583113"
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{|style="color:black" cellpadding="4" cellspacing="2" border="1" align="middle"
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! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''Sample ID'''</FONT>
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! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''Arabinose conc.'''</FONT>
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! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''Construct Type'''</FONT>
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|-
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|'''positive'''
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|0% wt
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|BFP
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|-
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|'''positive'''
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|0.2% wt
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|BFP
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|'''positive'''
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|0.5% wt
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|BFP
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|'''positive'''
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|1% wt
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|BFP
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|'''negative'''
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|0% wt
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|Top 10
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|'''negative'''
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|0.2% wt
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|Top 10
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|'''negative'''
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|0.5% wt
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|Top 10
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|'''negative'''
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|1% wt
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|Top 10
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<p>Unfortunately from this data we had to conclude that our device does not differ in blue fluorescence from the negative controls meaning thus: the Blue Fluorescent Protein is not properly folded and not functional.</p>
 
<p>Unfortunately from this data we had to conclude that our device does not differ in blue fluorescence from the negative controls meaning thus: the Blue Fluorescent Protein is not properly folded and not functional.</p>
  
 
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Revision as of 14:25, 18 September 2015

pAra + fusion protein BFP_Spycatcher_His


This device expresses the fusion protein BFP_SpyCatcher_His under arabinose control.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 131
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 71
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



This device expresses the fusion protein BFP_SpyCatcher_His under arabinose control.

It was created as a fusion protein from the following biobricks:



The SpyTag SpyCatcher couple reacts under physiological conditions spontaneously to form a covalent isopeptide bond between both proteins linking them irreversibly together.

CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG.

In our project we created Biobricks where we added specific tags to the CsgA protein in order to produce a functionalised amyloid nanowire. These modifications aim at increasing adhesive properties towards a certain surface or material. Examples are CsgA_His or the device CsgA_Hydroxyapatite affinity with hydroxyapatite being the main component of teeth.

With this device, we were planning to prove the possibility of modifying our amyloid nanowires with a functionality such as a fusion protein.

Unfortunately, due to a frameshift in our construct CsgA_SpyTag we were not able to produce amyloid nanowires with this certain tag in time.

Characterization

  • This part was characterised using a fluorescent assay to investigate proper folding of the BFP


    • Fluorescence assay

    Fig. 1: Fluorescence signal normalized by the number of cells (OD600) over the time of 18 hours.
    {|style="color:black" cellpadding="4" cellspacing="2" border="1" align="middle" ! style="background:#0084A7;"|'''Sample ID''' ! style="background:#0084A7;"|'''Arabinose conc.''' ! style="background:#0084A7;"|'''Construct Type''' |- |'''positive''' |0% wt |BFP |- |- |'''positive''' |0.2% wt |BFP |- |'''positive''' |0.5% wt |BFP |- |'''positive''' |1% wt |BFP |'''negative''' |0% wt |Top 10 |'''negative''' |0.2% wt |Top 10 |'''negative''' |0.5% wt |Top 10 |'''negative''' |1% wt |Top 10 |}

    Unfortunately from this data we had to conclude that our device does not differ in blue fluorescence from the negative controls meaning thus: the Blue Fluorescent Protein is not properly folded and not functional.