Team:Cambridge-JIC/Constructs/gal4 hap1gr H2RBmRFP

From 2014.igem.org

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<li> We'll try out 35s->GAL4->HAP1GR->H2BmRFP. </li>
<li> We'll try out 35s->GAL4->HAP1GR->H2BmRFP. </li>
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<li> Since H2BmRFP has not been tried in Mp, we'll also try 35s->H2BmRFP </li>
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<li> Since H2BmRFP has not been tried in Mp, we'll also try 35s->H2BmRFP. A positive control for lab technique will be 35s->Venus N7</li>
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<li> We'll also use FF's original plasmid to try 35s-Hap1GR-mRFP, as a control for the Hap1GR. I expect low transformation rate as in the original plasmid, we're using 35s as promoter for HygR selection marker.
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<li> Not sure whether 35s->Gal4 has been tried (check with BP), so 35s-Gal4-venus n7 is doable with BP's construct as a control </li>
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<li> As another control, we'll try 35s-Gal4-H2mRFP </li>
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Revision as of 14:39, 24 July 2014

Gal4 - Hap1GR - H2BmRFP

Design

Aim of the construct

  • We'll try out 35s->GAL4->HAP1GR->H2BmRFP.
  • Since H2BmRFP has not been tried in Mp, we'll also try 35s->H2BmRFP. A positive control for lab technique will be 35s->Venus N7
  • We'll also use FF's original plasmid to try 35s-Hap1GR-mRFP, as a control for the Hap1GR. I expect low transformation rate as in the original plasmid, we're using 35s as promoter for HygR selection marker.
  • Not sure whether 35s->Gal4 has been tried (check with BP), so 35s-Gal4-venus n7 is doable with BP's construct as a control
  • As another control, we'll try 35s-Gal4-H2mRFP

End goal plasmid

Experimentation (up to date)

PCR

Attempt 1:

Gibson

Attempt 1:
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E-Coli transformation

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Agrobacteria transformation

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Spore preparation

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Spore transformation

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Evaluation

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