Team:Copenhagen/Project/Experimental

From 2011.igem.org

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<li>Digest the parental template DNA (which is methylated) with Dpn1 (which cut methylated DNA only)</li>
<li>Digest the parental template DNA (which is methylated) with Dpn1 (which cut methylated DNA only)</li>
<li>Transform XL1-Blue competent cells with the mutated plasmid</li>
<li>Transform XL1-Blue competent cells with the mutated plasmid</li>
-
 
+
<li>Once you have a colony on your plate you take it out and place it in and overnight culture</li>
 +
<li>You perform a Miniprep on your culture to purify your amplified plasmid </li>
 +
<li>Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site</li>
 +
<li>Run the digest of the plasmid and hope to see a number of bands corresponding to the number of restriction sites in the plasmid backbone. In the latter case your mutations failed </li>
 +
<li>Get it sequenced</li>
</ul></p>
</ul></p>
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<ul>
<ul>
-
<li>Once you have a colony on your plate you take it out and place it in and overnight culture</li>
+
 
-
<li>You perform a Miniprep on your culture to purify your amplified plasmid </li>
+
-
<li>Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site</li>
+
-
<li>Run the digest of the plasmid and hope to see a number of bands corresponding to the number of restriction sites in the plasmid backbone. In the latter case your mutations failed </li>
+
<li>Add prefix and suffix containing primers for the CYP in question and amplify with PCR</li>
<li>Add prefix and suffix containing primers for the CYP in question and amplify with PCR</li>
<li>Purify with DNA Purification kit</li>
<li>Purify with DNA Purification kit</li>
<li>Cut with restrictionsenzymes EcoR1 and Pst1</li>
<li>Cut with restrictionsenzymes EcoR1 and Pst1</li>
-
<li>Run on a gel and cut the wanted band out </li>
+
<li>Heat shock to kill restrictionenzymes</li>
-
<li>Extract the DNA from the gel pieces by using a Gel Extraction kit</li>
+
<li>Cut the linarized plasmid backbone with Pst1 and EcoR1 </li>
<li>Cut the linarized plasmid backbone with Pst1 and EcoR1 </li>
<li>Ligate CYP and Plasmid </li>
<li>Ligate CYP and Plasmid </li>
<li>Transform in XL1-Blue</li>
<li>Transform in XL1-Blue</li>
<li>Overnight culture</li>
<li>Overnight culture</li>
-
<li>Hurra - You have a Biobrick</li>
+
<li>Mini Prep</li>
 +
<li>Get it sequenced</li>
 +
<li>Sent it to iGEM</li>
</ul></p>
</ul></p>
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<li>Cut the newly made Cyp BioBrick with restrictionsenzymes Xba and Pst1</li>
<li>Cut the newly made Cyp BioBrick with restrictionsenzymes Xba and Pst1</li>
-
<li>Run on a gel and cut the wanted band out </li>
+
<li>Heat Shock to kill the restriction enzymes </li>
<li>Put it in the freezer</li>
<li>Put it in the freezer</li>
-
<li>Cut the purified BioBrick J004500 ekspression vector from the kit with Spe1 and EcoR1 </li>
+
<li>Cut the purified BioBrick J004500 ekspression vector from the kit with Spe1 and Pst1 </li>
<li>Ligate CYP and Vector</li>
<li>Ligate CYP and Vector</li>
-
<li>Transform in XL1-Blue</li>
+
<li>Transform in BL21</li>
-
<li>Overnight culture</li>
+
 
-
<li>Hurra - You have a CyperMan</li>
+
<li>Grow cells O/N while inducing ekspression of CYP with IPTG.</li>
 +
<li>Take tests (1h, 2h, 4h, next day)  and prepare them for SDS page</li>
 +
<li>Run the SDS and hope to see increased amount of a band with the size of the CYP</li>
 +
<li>Make a membrane preparation to get micelles with cytochrome</li>
 +
<li><a href="https://2011.igem.org/Team:Copenhagen/Results" style="text-decoration:none; color:black;">CO measurements</a> for the membrane preperations</li>
 +
<li>Test the cytochromes ability to transform aminoacids to oximes with <a href="https://2011.igem.org/Team:Copenhagen/Results" style="text-decoration:none; color:black;">TLC</></li>
 +
<li>Kill Fungus</li>
</ul></p>
</ul></p>

Latest revision as of 14:31, 18 September 2011


How to
Mutate

  • Analysis have made you realise the need for mutations in the CYP in order to remove resctrictionsites
  • You design primers that fits your template but contains the altered base
  • You mix your primers with the template, dNTP, polymerase and HF buffer and run a non amplifying PCR
  • Digest the parental template DNA (which is methylated) with Dpn1 (which cut methylated DNA only)
  • Transform XL1-Blue competent cells with the mutated plasmid
  • Once you have a colony on your plate you take it out and place it in and overnight culture
  • You perform a Miniprep on your culture to purify your amplified plasmid
  • Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site
  • Run the digest of the plasmid and hope to see a number of bands corresponding to the number of restriction sites in the plasmid backbone. In the latter case your mutations failed
  • Get it sequenced
How to
make a BioBrick

  • Add prefix and suffix containing primers for the CYP in question and amplify with PCR
  • Purify with DNA Purification kit
  • Cut with restrictionsenzymes EcoR1 and Pst1
  • Heat shock to kill restrictionenzymes
  • Cut the linarized plasmid backbone with Pst1 and EcoR1
  • Ligate CYP and Plasmid
  • Transform in XL1-Blue
  • Overnight culture
  • Mini Prep
  • Get it sequenced
  • Sent it to iGEM
How to
make a CyperMan

  • Cut the newly made Cyp BioBrick with restrictionsenzymes Xba and Pst1
  • Heat Shock to kill the restriction enzymes
  • Put it in the freezer
  • Cut the purified BioBrick J004500 ekspression vector from the kit with Spe1 and Pst1
  • Ligate CYP and Vector
  • Transform in BL21
  • Grow cells O/N while inducing ekspression of CYP with IPTG.
  • Take tests (1h, 2h, 4h, next day) and prepare them for SDS page
  • Run the SDS and hope to see increased amount of a band with the size of the CYP
  • Make a membrane preparation to get micelles with cytochrome
  • CO measurements for the membrane preperations
  • Test the cytochromes ability to transform aminoacids to oximes with TLC
  • Kill Fungus


Comments or questions to the team? Please mail us at igemcopenhagen@gmail.com

Retrieved from "http://2011.igem.org/Team:Copenhagen/Project/Experimental"