Brown: Team Resistance/2 July 2008


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2 July 2008

In order to make our SRRz gene cassette bio-brick compatible, we wanted to PCR our DNA. In the Jain/Mekalanos paper, they inserted a 1.5kb insert into the pBAD18 plasmid using EcoRI and HindII sites. Because HindII is a blunt end restriction enzyme, we can’t cut out the same 1.5kb portion. We found the sequence of the SRRz gene cassette on NCBI to be 1.3kb. We designed primers to amplify just the 1.3kb cassette, so as to leave out the extra 200bp in Harvard’s insert.

PCR of pVJ4 plasmid (pBAD+SRRz) Need to have:

  • dNTP mixes
  • polymerase (in freezer)
  • MgCl2 (dry stock)

Found taq-PCR kit in freezer from last year’s team

Designed primers based off of paper referenced in the Jain Mekalanos paper concerning the pBAD18 plasmid. In the paper (Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter Guzman, L.-M., D. Belin, M. J. Carson, and J. Beckwith) three primers to be used with the pBAD18 promoter were specified: 5’: CTGTTTCTCCATACCCGTT 3’(1): CTCATCCGCCAAAACAG 3’(2): GGCTGAAAATCTTCTCT

For melting points of primers: 2degrees C for every A and T 4degrees C for every C and G 5’: 56degrees C 3’(1): 52degrees C 3’(2): 48degrees C

Reaction 1: 5’/3’(1) → 50degrees C Reaction 2: 5’/3’(2) → 46degrees C

Dilutions: Primer 1: 5’pBAD Primer 2: 3’1pBAD Primer 3: 3’2pBAD

Primer 1: 25.95nmoles x2= 51.86ul H20 Primer 2: 46.01nmoles x2= 92.02ul H20 Primer 3: 43.09nmoles x2= 86.18ul H20

  • This gives a 500uM concentration of the primers but we want a 20uM concentration
  • To create a 20uM concentration: 1ul in 24ul H20 for each primer

DNA from miniprep: 10.7ng/ul Dilute to 1ng/mL in the final mix (consists of 50ul) Dilute DNA to 1ul in 10ul H20 (and then add this dilution of DNA to the PCR mix)

Mastermix rotocol from Adrian

Create master mix without primers so that it can be re-used Add taq-polymerase last

Tested function of pDKL02 plasmid lysis (obtained from Harvard, it contains SRRz cassette under control of IPTG-inducible promoter)

OD (pre-dilution) pDKL02 col1: 0.093 col2: 0.106 pVJ4 col1: 0.331 col2: 0.211

From colony 1 of pDKL02, and colony 1 of pVJ4, made 3, 1:100 dilutions and allowed them to reach mid-log phase (6 dilutions total) Dilution 1: add .2% arabinose Dilution 2: add .2% IPTG Dilution 3: control

Initial OD measurements: pDKL02: +IPTG: 0.019 +arabinose: 0.010 control: 0.018 (growing too slowly→ also need to grow these cells in media with lactose for IPTG induction to function)

pVJ4: +IPTG: 0.070 +arabinose: 0.061 control: 0.054 Added respective substances to respective pVJ4 cultures