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From 2012.igem.org

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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!!!!