DNA ScienceDay 1

Amplifying and Cutting

Physical Biology Bootcamp

October 2006

Caltech

Being Quantitative AboutGene Expression

• We can now answer a variety of questions quantitatively:

Setty et al. (2003)

Small et al. (1992, 1996)Elowitz et al. (2000)

How much? When? Where?

• Quantitative data demands quantitative models! In vivo modeling is needed.

The lac Operon, Where it all Began

• Modularity: Once the toolbox is developed you just need to shuffle the motifs to obtain novel behavior.

The Players of the lac Operon

So, what’s a plasmid?

Quantifying gene expression

• Many ways of measuring gene expression– LacZ activity– GFP fluorescense– mRNA level

Tom Kuhlman and Terry Hwa

• Does the message depend on the messenger?– Are the different reporters linear with respect to each

other?

What are the tools?

• PCR = Xerox Machine– Amplify DNA

• Restriction Enzymes = Scissors– Target very specific DNA

sequences

• Ligase = Glue

• Transformation and DNA extraction

Making a modular plasmid

Lutz and Bujard (1997)

• Copy number from 3 to 70 per cell

• Four possible antibiotic resistances

• Four promoters with three different inducers

/HindIII

5 PlacUV5

The big picture

• Extract the lacZ gene from plasmid pZE21-lacZ (I extracted it originally from wild type type E. coli: MG1655, GenBank U00096).

• Put it into a pZS25 vector– pSC101 origin of replication, ~10 copies.– Kanamycin resistance– PlacUV5, repressed by the Lac repressor and

induced by IPTG.

• Measure and compare the induction!

Doing a Restriction Digest

• Lambda DNA (NC_001416) predigested by HindIII.– Show in Vector NTI.– Go to the NEB site.– We’ll digest it with EcoRI.

• Obtain our vector by digesting pZS25 plasmid with KpnI and HindIII.– Show in Vector NTI– Different looping lengths and sequences.

• Run the results on an agarose gel.– Analyze our results.– Extract certain DNA fragments (the vector).

Digestion Protocol

• Lambda/HindIII:– 3 ul Lambda/HindIII (1.5 ug)

5 ul NEBuffer EcoRI (10x)40 ul ddH2O2 ul EcoRI50 ul Total

• Double Digest:– Start the cloning with ~5 ug of your vector– Just ~300 ng of DNA for the controls

• Let sit for 2-3 hours at 37ºC

Polymerase Chain Reaction

Polymerase Chain Reaction

• Designing a primer– Adding a couple of sites– (APh162 Primer.doc)

• The components and protocol– (Accuprime II.pdf)

• Draw cycle!– 1. 94C for 2 min – DNAP activation

2. 94C for 15 s – melting3. 60C for 30 s – anheling4. 68C for 3.5 min (min/kb) – elongation5. Go back to 2 for a total of 35 cycles6. Store at 4C

Gel Electrophoresis

• Preparing the samples:– <150 ng– Loading dye– DNA ladders (pg. 10)

• Run 1% TAE gel at 90 V for ~80 min

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