Team:IvyTech-South Bend/Notebook

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Latest revision as of 03:00, 4 October 2012

Notebook

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Lab Notebook: See our weekly lab work

June 18, 2012 Week 1. Strategizing and preparing presentation of project summery to Ivy Tech advisory board. Parts research and descriptions.

June 25, 2012 Week 2. Continuation of parts research including promoters & killswitch screening and selection. Constitutive PTeT promoter, RBS, double terminator.

July 2, 2012 Week 3. Checked stock of E.coli cultures and poured plates. Continuation of parts research and construction. Began pulling parts from registry and transformed into electro-competent E. Coli cells

July 9, 2012 Week 4. Oops Event!!! Protocol correction due to low yield on transformation. Kill-switch transformation was redone (serial dilution test for transformation efficiency) Kanamycin proves to be better selection for transformation for the psb1Ak3 plasmid backbone.

Pulled K190015 because its an arsenic sensitive promoter with RFP built into the plasmid

Transformed K190015 onto Psb1A13 in E.coli

July 16, 2012 Week 5. Transferred to Arsenic inducible promoter J33201 with Ars R repressor binding site. To be able to test for decreasing sensitivity to Arsenic.

July 23, 2012 Week 6. Re did transformation on electro competent E.Coli cells. Results yielded pink & white colonies. Expanded the colonies and purified K190015 DNA

-PINK Hypothesized to be successful transformation with RFP

-WHITE Unsure, proceeded to test in wk. 7

August 1, 2012 Week 7. To test sensitivity of k190015; including pink and white colonies, ran 96 well and stimulating with dilute Arsenic sol. Results were inconclusive.

August 13, 2012 Week 9. Ran gel to confirm band length of suspected parts, reran the 96 well plates yielding better results.

August 20, 2012 Week 10. To confirm j33201 working on psb1k3 and selected on LB/Kan plates, retransform k190015 GFP with E0840 as a reporter

August 27, 2012 Week 11. Extract K190015 DNA, add Transform. Repeat 3 times and plate. The plates we poured were less than 10% red, the rest were white. Total of 64hrs. for growth to appear.

September 3, 2012 Week 12.

Begin Construction of K935001 – which is part J33201 with a double terminator B0015. Restrictions and Ligations selecting over LB Tet plates

No Growth on Tet plates.

September 10, 2012 Week 13.

Running Gel to confirm positive transformations and plasmid exchange between J33201 and K190015 Began reviving E. Coli biosensor project and pulled part K11702 because of its quorum sensing detection sensitive promoter. Transformed in E.Coli on a psB1AT3 backbone and selected on LB Amp plates.

September 17, 2012 Week 14.

Redid transformation of J33201 with double terminator B0015 and selecting on LB Amp plates. SUCCESS!! K935001 Lives!! Positive transformation results Extract DNA and get ready to build.

September 24, 2012 Week 15.

Begin construction of K935002, K935003, K935004 and K935005. All of these parts will contain K935001 to contain multiple pArs - ArsR There is NO RFP!!!! Using exchange of K190015 and J33201 (J33201 on J61002 plasmid with RFP) now know as K935006, to redo ligations to include RFP where K19005 was not used.

Extract DNA from all new transformations and Nano Drop! Begin building in to Chloramphenicol backbones

October 1, 2012 Week 16

Extract DNA from previously non RFP’d parts and restrict and ligate again to include RFP SUCCESS!! Great Nano drop results. WE HAVE DNA!!! All except K935003. Still contains J33201 with double terminator without RFP. Redo transformation and make a NEW and improved part just like K935003 but now with RFP = K935004!! Spin Down, Spin Down, Spin Down and extract!!!!

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-You are provided with this team page template with which to start the iGEM season.  You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki.  You can find some examples <a href="https://2008.igem.org/Help:Template/Examples">HERE</a>.
+</a>
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-<h2 class="title"><a href="#">Lab Notebook: See our weekly lab work</a></h2>
+<h2 class="title">Lab Notebook: See our weekly lab work</h2>
-June 18, 2012  Week 1.
+'''''June 18, 2012  Week 1.'''''
-Staging and presentation of project storming to Ivy Tech advisory board.  Parts research and descriptions.
+Strategizing  and preparing presentation of project summery  to Ivy Tech advisory board.  Parts research and descriptions.
-June 25, 2012  Week 2.
-Continuation of parts research including promoters & killswitch screening and selction. Constitutive PTeT promoter, RBS, double terminator.
-July 2, 2012   Week 3.
-Checked cultures and paired plates.
-Continuation of parts research and construction
-July 9, 2012   Week 4.
+'''June 25, 2012  Week 2.'''
-Oops Event!!!  Protocol correction due to low yield or transformation.  Killswitch transformation was redone (serial dilution test for transformation efficiency) Kamamiosin proves to be better selection for transformation for the psb1Ak3 plasmid backbone.
+Continuation of parts research including promoters & killswitch screening and selection. Constitutive PTeT promoter, RBS, double terminator.
-Pulled K190015 because its an arsenic sensitive promoter ẅ RFP
-Transformed K190015 on Psb1A13
-July 16, 2012  Week 5.
+'''July 2, 2012   Week 3.'''
-Propped Test promoter and transferred to Arsenic inducible promoter J33201 with Ars R repressor binding site. To be able to test for decreasing sensitivity  to Arsenic.
+Checked stock of E.coli cultures and poured plates.
+Continuation of parts research and construction.  Began pulling parts from registry and transformed into electro-competent E. Coli cells
-July 23, 2012  Week 6.
-Redid transformation on electrocompetent eColi cells.  Results yielded pink & white colonies. Expanded the colonies and purfied K190015
--PINK  Hypothesized to be succesful transformation with Rtt
+'''July 9, 2012   Week 4.'''
+Oops Event!!!  Protocol correction due to low yield on transformation.  Kill-switch transformation was redone (serial dilution test for transformation efficiency) Kanamycin proves to be better selection for transformation for the psb1Ak3 plasmid backbone.
+Pulled K190015 because its an arsenic sensitive promoter with RFP built into the plasmid
+Transformed K190015 onto Psb1A13 in E.coli
+'''July 16, 2012  Week 5.'''
+Transferred to Arsenic inducible promoter J33201 with Ars R repressor binding site. To be able to test for decreasing sensitivity  to Arsenic.
+'''July 23, 2012  Week 6.'''
+Re did transformation on electro competent E.Coli cells.  Results yielded pink & white colonies. Expanded the colonies and purified K190015 DNA
+-PINK  Hypothesized to be successful transformation with RFP
 -WHITE  Unsure, proceeded to test in wk. 7
-August 1, 2012  Week 7.
+'''August 1, 2012  Week 7.'''
 To test sensitivity of k190015; including pink and white colonies, ran 96 well and stimulating with dilute Arsenic sol. Results were inconclusive.
-August 13, 2012  Week 9.
+'''August 13, 2012  Week 9.'''
 Ran gel to confirm band length of suspected parts, reran the 96 well plates yielding better results.
-August 20, 2012  Week 10.
-To confirm S33201
-August27, 2012   Week 11.
+'''August 20, 2012  Week 10.'''
-Extract K190015 10ul, add DNA to ecom cells(5ul) into a tube, add to cuvette and flick to micropulsar.  Take reading from BioRad and add soc immediately. 950ul of soc into cuvette, extract all contents and place into tubes.  Repeat 3 times.  Put in incubator for 1 hr@30°C, make 3 serial dilutions.  Once serial dilution is complete spread on the amp plates, put streaked plates in incubator.
+To confirm j33201 working on psb1k3 and selected on LB/Kan plates, retransform k190015 GFP with E0840 as a reporter
-The plates we poured were less than 10% red, the rest were white.
+'''August 27, 2012   Week 11.'''
+Extract K190015 DNA, add Transform.  Repeat 3 times and plate.  The plates we poured were less than 10% red, the rest were white.
 Total of 64hrs. for growth to appear.
+'''September 3, 2012   Week 12.'''
+Begin Construction of K935001 – which is part J33201 with a double terminator B0015.  Restrictions and Ligations selecting over LB Tet plates
+No Growth on Tet plates.
+'''September 10, 2012 Week 13.'''
+Running Gel to confirm positive transformations and plasmid exchange between J33201 and K190015
+Began reviving E. Coli biosensor project and pulled part K11702 because of its quorum sensing detection sensitive promoter.  Transformed in E.Coli  on a psB1AT3 backbone and selected on LB Amp plates.
+'''September 17, 2012 Week 14.'''
+Redid transformation of J33201 with double terminator B0015 and selecting on LB Amp plates.
+SUCCESS!! K935001 Lives!! Positive transformation results
+Extract DNA and get ready to build.
+'''September 24, 2012 Week 15.'''
+Begin construction of K935002, K935003, K935004 and K935005.  All of these parts will contain K935001 to contain multiple pArs - ArsR
+There is NO RFP!!!! Using exchange of K190015 and J33201 (J33201 on J61002 plasmid with RFP) now know as K935006, to redo ligations to include RFP where K19005 was not used.
+Extract DNA from all new transformations and Nano Drop!
+Begin building in to Chloramphenicol backbones
+'''October 1, 2012 Week 16'''
+Extract DNA from previously non RFP’d parts and restrict and ligate again to include RFP
+SUCCESS!! Great Nano drop results.  WE HAVE DNA!!!
+All except K935003.  Still contains J33201 with double terminator without RFP.
+Redo transformation and make a NEW and improved part just like K935003 but now with RFP = K935004!!
+Spin Down, Spin Down,  Spin Down and extract!!!!