IGEM:MIT/2005/Receiver Head Unit: scFv: Difference between revisions

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==POC==
==POC==
Jenny
Alias: Jenny<BR>
Location(s): BUG Lounge, Kate's Lab<BR>
E-mail: JennyN@mit.edu<BR><BR>
 
[[../Experiments#Experiment Status/|This is what I'm working on currently.]]
 
==Function==
==Function==
*To provide a landing site for ligand.
*To provide a landing site for ligand.
*To cause dimerization/conformational change in the Receiver.
*To cause dimerization/conformational change in the Receiver.
*To transmit signal to Receiver.
*To transmit signal to Receiver.
*Will use anti-fluorescein scFv for test phase.
*Ultimately use anti-rabbit scFv for system.
==Device Depiction==
==Device Depiction==
<gallery>
<gallery>
[[Image:ScFv.jpg|thumb|100px|scFv]]
Image:ScFv.jpg|scFv
</gallery>
</gallery>


==Device Parts==
==Device Parts==
==Current Status==
==Current Status==
[[../Experiments#Jenny/|Experiments Status]]
<b>FWD for Testing scFvs using anti-FLAG</b>
CATCAT GAATTC GCGGCCGC T TCTAGA TG GACGTCGTTATGACTCAAACACC
-rand- -EcoRI --NotI--  -XbaI[---]      Primer
                              start
*Primers have been designed for biobrick format.
*Primers have been designed for biobrick format.
*pCT-4-4-20, pCT302 S101A, pCT-4M5.3 anti-fluorescein plasmids miniprepped
*pCT-4-4-20, pCT302 S101A, pCT-4M5.3 anti-fluorescein plasmids miniprepped


  FWD: 5' CATCAT GAATTC TCTAGA GACGTCGTTATGACTCAAAC 3'
  FWD: 5' CATCAT GAATTC GCGGCCGC T TCTAGA GACGTCGTTATGACTCAAACACC 3'
                EcoRI  XbaI      Primer
          rand  EcoRI  NotI      XbaI      Primer


  REV: 5' GCTCAT CTGCAG ACTAGT TTATTA CTTATCATCATCATCCTTATAATC GGAGACGGTGACTGAGGTTCC 3'
  REV: 5' GCTCAT CTGCAG GCGGCCGC T ACTAGT TTATTA CTTATCATCATCATCCTTATAATC GGAGACGGTGACTGAGGTTCC 3'
                PstI  SpeI  Stop          FLAG Tag                Primer
          rand  PstI  NotI      SpeI  Stop          FLAG Tag                Primer
 
==Experiments==
*To test whether the scFvs are expressed? How?
*We need constitutive promoter with RBS upstream of it. There is one in the library available. Biobrick scFv onto RBS/promoter, and it should express. Do a western blot to see if protein expression by using FLAG tag. We need anti-FLAG antibody (ask Natalie).
 
[[../Completed Work/]]


==Open Issues/Questions==
==Open Issues/Questions==
*<S>TAG (myc) to know that the fluorescein has bound.</s>
*<s>How to design primers so that the sequence is in frame?</s>


==Need Help With==
==Additional Information==
Check to see if primers are correct. Will talk to Kate 7/13.
 
<b> Anti-rabbit </b>
: Sequence: [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=48727569 Sequence of synthetic construct anti-rabbit IgG scFV mRNA, partial cds]
: Paper: [http://pubs.acs.org/cgi-bin/article.cgi/ancham/2005/77/i03/pdf/ac048655w.pdf scFv Antibody Piezoimmunosensors]
: Summary: [[../Summary scFvSensor/]]

Latest revision as of 22:33, 14 August 2005

POC

Alias: Jenny
Location(s): BUG Lounge, Kate's Lab
E-mail: JennyN@mit.edu

[[../Experiments#Experiment Status/|This is what I'm working on currently.]]

Function

  • To provide a landing site for ligand.
  • To cause dimerization/conformational change in the Receiver.
  • To transmit signal to Receiver.
  • Will use anti-fluorescein scFv for test phase.
  • Ultimately use anti-rabbit scFv for system.

Device Depiction

  • scFv

    scFv

Device Parts

Current Status

[[../Experiments#Jenny/|Experiments Status]]

FWD for Testing scFvs using anti-FLAG
CATCAT GAATTC GCGGCCGC T TCTAGA TG GACGTCGTTATGACTCAAACACC
-rand- -EcoRI --NotI--   -XbaI[---]      Primer
                              start
  • Primers have been designed for biobrick format.
  • pCT-4-4-20, pCT302 S101A, pCT-4M5.3 anti-fluorescein plasmids miniprepped
FWD: 5' CATCAT GAATTC GCGGCCGC T TCTAGA GACGTCGTTATGACTCAAACACC 3'
         rand   EcoRI   NotI      XbaI       Primer

REV: 5' GCTCAT CTGCAG GCGGCCGC T ACTAGT TTATTA CTTATCATCATCATCCTTATAATC GGAGACGGTGACTGAGGTTCC 3'
         rand   PstI   NotI       SpeI   Stop          FLAG Tag                 Primer

Experiments

  • To test whether the scFvs are expressed? How?
  • We need constitutive promoter with RBS upstream of it. There is one in the library available. Biobrick scFv onto RBS/promoter, and it should express. Do a western blot to see if protein expression by using FLAG tag. We need anti-FLAG antibody (ask Natalie).

[[../Completed Work/]]

Open Issues/Questions

Additional Information

Anti-rabbit

Sequence: Sequence of synthetic construct anti-rabbit IgG scFV mRNA, partial cds
Paper: scFv Antibody Piezoimmunosensors
Summary: [[../Summary scFvSensor/]]
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