Team:Caltech/Notebook/Proteorhodopsin

From 2012.igem.org

(Difference between revisions)
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Julia - transformed K572005 (proteorhodopsin gene)
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Julia - transformed K572005 (proteorhodopsin gene from the Parts Registry)
<br>
<br>
Daisy - ordered primers for Polymerase Cycling Assembly (PCA) reaction to create a proteorhodopsin optimized for E. coli
Daisy - ordered primers for Polymerase Cycling Assembly (PCA) reaction to create a proteorhodopsin optimized for E. coli
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<br>
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<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
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<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
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Chenxi - Transformed pKD46 into wild type MG1655 E.coli strain using electroporation; designed primers to PCR pKD4 Kan resistance gene for lambda red knockout of nuo and ndh genes (NADH dehydrogenases)
Chenxi - Transformed pKD46 into wild type MG1655 E.coli strain using electroporation; designed primers to PCR pKD4 Kan resistance gene for lambda red knockout of nuo and ndh genes (NADH dehydrogenases)
<br>
<br>
-
Julia - sequenced K572005
+
Julia - miniprepped K572005 and sent it in for sequencing
 +
<br>
 +
Daisy - made 50 uL aliquots of electrocompetent E. coli cells
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
<br>
<br>
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-
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
 
<br>
<br>
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<font size="+2"><a name="6_26_12">6/26/12</a></font>
<font size="+2"><a name="6_26_12">6/26/12</a></font>
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Chenxi - PCR pKD4 kan for nuo and ndh; ran gel
Chenxi - PCR pKD4 kan for nuo and ndh; ran gel
<br>
<br>
-
 
+
Julia - K572005 sequence is completely different from the one listed in the Parts Registry, but the sequence matches with a different part (contamination)
-
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
<br>
<br>
-
 
-
 
<font size="+2"><a name="6_27_12">6/27/12</a></font>
<font size="+2"><a name="6_27_12">6/27/12</a></font>
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<br>
<br>
<br>
-
 
+
Daisy - began PCA protocol: ran PCR to make the preassembly mix, IPIPE, and VPIPE constructs; ran gel electrophoresis on the PCR products to check that the PCRs worked
-
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
<br>
<br>
-
 
-
 
<font size="+2"><a name="6_28_12">6/28/12</a></font>
<font size="+2"><a name="6_28_12">6/28/12</a></font>
<br>
<br>
<br>
<br>
-
 
+
Daisy - continued PCA protocol: ran PCR to make the CPEC and IPIPE (from existing IPIPE, not preassembly mix) constructs; transformed electrocompetent cells with CPEC+VPIPE (positive control; no change the protocol), CPEC-VPIPE (negative control; replaced VPIPE with sterile water), IPIPE+VPIPE (alternate positive control), and VPIPE (alternate negative control) and plated them
-
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
<br>
<br>
-
 
-
 
<font size="+2"><a name="6_29_12">6/29/12</a></font>
<font size="+2"><a name="6_29_12">6/29/12</a></font>
<br>
<br>
<br>
<br>
-
 
+
Daisy - plate results: VPIPE and CPEC-VPIPE negative controls had no colonies, IPIPE+VPIPE had only one colony, CPEC+VPIPE had many colonies; started overnight cultures of IPIPE+VPIPE and CPEC+VPIPE constructs of proteorhodopsin
-
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
<br>
<br>
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<br>
<br>
<br>
<br>
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<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
Daisy - miniprepped the overnight cultures and measured their concentrations
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
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<br>
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<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
 
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
<br>
<br>
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<br>
<br>
<br>
<br>
-
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
Daisy - sent miniprep samples in for sequencing
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
<br>
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<br>
<br>
<br>
<br>
-
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Proteorhodopsin#Calendar">Back to Proteorhodopsin Notebook Calendar</a>
+
Daisy - sequencing results returned: IPIPE+VPIPE construct is incorrect; accidentally mixed the CPEC+VPIPE sequences (will redo)
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
 +
<br>
 +
 
 +
<font size="+2"><a name="7_4_12">7/4/12</a></font>
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<br>
 +
<br>
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Daisy - regrew 3 CPEC+VPIPE overnight cultures and made glycerol stocks
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
 +
<br>
 +
 
 +
<font size="+2"><a name="7_5_12">7/5/12</a></font>
 +
<br>
 +
<br>
 +
Daisy - miniprepped the CPEC+VPIPE cultures, measured their concentrations, and sent them in for sequencing
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
 +
<br>
 +
 
 +
<font size="+2"><a name="7_6_12">7/6/12</a></font>
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<br>
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<br>
 +
Daisy -
 +
<br>
 +
<a a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/Degradation#Calendar">Back to the top</a>
 +
<br>
<br>
<br>

Revision as of 17:11, 2 October 2012



Proteorhodopsin Notebook


6/22/12

Julia - transformed K572005 (proteorhodopsin gene from the Parts Registry)
Daisy - ordered primers for Polymerase Cycling Assembly (PCA) reaction to create a proteorhodopsin optimized for E. coli
Back to the top

6/25/12

Chenxi - Transformed pKD46 into wild type MG1655 E.coli strain using electroporation; designed primers to PCR pKD4 Kan resistance gene for lambda red knockout of nuo and ndh genes (NADH dehydrogenases)
Julia - miniprepped K572005 and sent it in for sequencing
Daisy - made 50 uL aliquots of electrocompetent E. coli cells
Back to the top

6/26/12

Chenxi - PCR pKD4 kan for nuo and ndh; ran gel
Julia - K572005 sequence is completely different from the one listed in the Parts Registry, but the sequence matches with a different part (contamination)
Back to the top

6/27/12

Daisy - began PCA protocol: ran PCR to make the preassembly mix, IPIPE, and VPIPE constructs; ran gel electrophoresis on the PCR products to check that the PCRs worked
Back to the top

6/28/12

Daisy - continued PCA protocol: ran PCR to make the CPEC and IPIPE (from existing IPIPE, not preassembly mix) constructs; transformed electrocompetent cells with CPEC+VPIPE (positive control; no change the protocol), CPEC-VPIPE (negative control; replaced VPIPE with sterile water), IPIPE+VPIPE (alternate positive control), and VPIPE (alternate negative control) and plated them
Back to the top

6/29/12

Daisy - plate results: VPIPE and CPEC-VPIPE negative controls had no colonies, IPIPE+VPIPE had only one colony, CPEC+VPIPE had many colonies; started overnight cultures of IPIPE+VPIPE and CPEC+VPIPE constructs of proteorhodopsin
Back to the top

6/30/12

Daisy - miniprepped the overnight cultures and measured their concentrations
Back to the top

7/1/12


Back to the top

7/2/12

Daisy - sent miniprep samples in for sequencing
Back to the top

7/3/12

Daisy - sequencing results returned: IPIPE+VPIPE construct is incorrect; accidentally mixed the CPEC+VPIPE sequences (will redo)
Back to the top

7/4/12

Daisy - regrew 3 CPEC+VPIPE overnight cultures and made glycerol stocks
Back to the top

7/5/12

Daisy - miniprepped the CPEC+VPIPE cultures, measured their concentrations, and sent them in for sequencing
Back to the top

7/6/12

Daisy -
Back to the top

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