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From 2013.igem.org

Characterization Notebook

Goal: Develop a well-characterized library of parts for the MoClo Assembly Method

Subgoals:

• Assemble level 0 parts needed
• Constitutive Promoter Characterization Project:
• J23100_AB, J23113_AB, J23115_AB, J23115_EB, J23116_AB, J23116_EB, J23117_AB, J23118_AB, J23119_AB
• BCD1_BC, BCD2_BC, BCD8_BC, BCD12_BC, BCD13_BC, BCD16_BC: completed 6/27/13
• Repressible Promoter Characterization Project:
• C0012 (LacI), C0062_CD (LuxR), C0071_CD (rhlR_CD), pLacI (R0010_FB)
• Assemble level 1 circuits for characterization of Constitutive Promoter Library
• 160 circuits that have different combinations of J231XX promoters and RBS’s
• 1 set with all promoters + BCD2
• 1 set with all RBS’s+J23100
• Assemble level 1 circuits for characterization of Repressible Promoter
• Run flow cytometry on the promoter characterization circuits
• Assemble level 2 inverter as an example of the library’s use
• Switch the backbone in some existing level 1 parts to study how different antibiotics affect the flow cytometry data: completed:6/21/13

May

Week of May 12, 2013

• Learned about synthetic biology, MoClo, and developed plans for our summer projects, specifically histidine kinase and quorum sensing.
• Learned about Clotho software suite and learned how to use Eugene, RavenCAD, and Pigeon from other members of the CIDAR lab.
• Familiarized ourselves with protocols and lab etiquette
• Stocked up on lab supplies (plates, broth, plasmids)

Week of May 19, 2013

• Began to create new bicistronic design RBS’s (BCDs): used PCR on BCD2 template to make BCD1, followed with LV0 reaction
• Began to restock low concentration and low volume plasmid for level 0 parts

Week of May 26, 2013

• Sent BCD1 made last week for sequence verification
• Assembled Lv1 circuits to test out new fluorescent proteins (CyPet, DsRed, mCitrine, mOrange) and for flow cytometry training
• Ran flow cytometry with Traci for our training
• Continued to restock low concentration and low volume parts