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Assignments Due

• Go to the Presentation Schedule
  • Presenters see the guidelines for Journal Club Presentation
  • Everyone else (undergraduate and graduate students) read listed articles on Simple control and be prepared for discussion

Overview of Experiment

Today you will begin building your new circuit. Lab sessions 5-8 are self-scheduled (lab "begins"after the presentations and "ends" at 6 p.m.) ; if you need to come in at other times (9:30 a.m. - 5:30 p.m., weekdays only!) / days to pull plates or start overnight cultures, please let me know so I can unlock the labs.

Before beginning procedures on Day 5, record an outline of your planned experimental strategy for building the simple circuit (described below) in your lab notebook. Include details such as restriction enzymes you will use and how you will confirm the identity of your construct (Hint: we identified transformants TWO different ways in lab) as well as expected results.

• You will be given mini preps of plasmid DNA for these THREE parts from the Registry
  • BBa_R0040 promoter/tetR, negative (part = 54 bp) [in pSB1A2, AmpR]
    
    
    
  • BBa_Q04400 TetR inverter with strong RBS (part = 902 bp) [in pSB2K3, Kanamycin = KanR]
    
    
    
  • BBa_E0840 GFP reporter with strong RBS (part = 878 bp) [in pSB1A2, AmpR]
    
  
  
• Use BioBrick Standard Assembly to build your construct
  • Restriction sites for EcoRI-HF, XbaI, SpeI, and PstI flank the BioBrick: ~ -E-X-[ ]-S-P- ~
  • Recall that one double digest will be used to cut out the insert from one plasmid and a second double digest will be used to open up the other plasmid; the insert and vector must be gel purified before ligating
  • You must decide which enzymes to use for each BioBrick in preparation for the ligations described below
    (If you really get "stuck" trying to choose the pairs of enzymes for each digest, come talk to me)
  
  
• Perform TWO ligations, in the order specified, to build the desired circuit
  • Ligation #1: R0040 + Q04400
    
    Colony PCR Screen: MCS_Fwd/MCS_Rev primers, looking for a 1194 bp band
    
    
  • Ligation #2: [R0040+Q04400] + E0840
    
    Colony PCR Screen: MCS_Fwd/MCS_Rev, looking for a ~ 2 kb band

Plasmid DNA mini preps and restriction enzyme digests are highly recommended to screen putative transformants: pick isolated colonies and grow overnight in 4 ml LB + Amp; use 3 ml for mini prep and make glycerol stock of 1 ml. If you decide to screen by colony PCR, you do not need a (+) control for PCR; however, you should run a (-) control reaction. 

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 4 November 2009