Difference between revisions of "Team:Warwick/Project2"

Line 36: Line 36:
 
<H3>Experiment 1: Establishing Anchor Protein</H3>
 
<H3>Experiment 1: Establishing Anchor Protein</H3>
  
−
<br>1. Golden gate assembly of plasmid backbones http://synbio.tsl.ac.uk/golden-gate-assembly-protocol/  
+
<br>1. Take a briobrick part (JO4450) and transform it into a chemically competent cell (Top 10)
−
and 4 G-blocks (one of each candidate anchor protein and Zif268)
+
<br>2. Grow cells overnight then miniprep them to extract the transformed plasmid
−
<br>Waiting on enzymes (hopefully Friday/ Monday)
+
<br>3. Double digestion of plasmid to cut out the RFP gene, leaving only the plasmid backbone
−
<br>Lpp-OMPa is done
+
<br>4. PCR the full construct G-Block (containing Zif268 and Lpp_OmpA) and digest the product with the corresponding enzymes
−
<br>BclA, Pgsa, (INP) - waiting on enzymes to cut out anchor proteins
+
<br>5. <a href="http://synbio.tsl.ac.uk/golden-gate-assembly-protocol/">Golden gate assembly</a> of plasmid backbones 
−
 
+
and full construct.
−
<br>2. Transform electrocompetent E. Coli cells with plasmid
+
<br>6. Transform electrocompetent E. <i>coli</i> cells with full plasmid
−
<br>NB - IPTG needs to be added
+
<br>7.Grow cells
−
<br>Grow cells
+
<br>8. Miniprep full construct plasmid from modified cells
−
<br>Prepared for microscopy (mounting medium)
+
<br>9. Digest plasmid with enzymes matching the cut sites around the Lpp_OmpA gene
−
<br>DNA on glass (waiting epoxy, aminated oligo, fluorescent marker) - Friday/Monday
+
<br>10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
−
 
+
<br>11. Ligate the anchor proteins into digested full construct plasmid
−
<br>3. Bind antibodies to flag tag - measure using fluorescent detection mechanism
+
<br>12. Transform all plasmids into electrocompetent E. <i>coli</i> - End product is 4 different types of cell, each expressing the full construct with slightly different transmembrane domains
−
<br>Antonia has components
+
<br>13. Prepare transformed cells for microscopy (mount cells on glass slides)  
−
 
+
<br>14. Bind antibodies to FLAG tag - measure under fluorescent microscope
−
<br>4. Compare intensities - select anchor protein that fluoresces brightest
+
<br>15. Determine whether the gene is being expressed and whether the expressed protein is being transported and is binding to the membrane of the E. <i>coli</i>
−
 
+
<br>16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
−
<br>Back-up:
+
−
<br>Lyse cells to see if expression is taking place
+
 
</p>
 
</p>
 
<p>
 
<p>

Revision as of 13:34, 9 September 2015

Warwick iGEM 2015

Open Menu