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From 2011.igem.org

Notebook

Week 1

PCR primers designed according to Biobrick specifications and ordered for rsaA C-terminal and WT esp. Because of the discrepancy in GC content between Staphylococcus epidermidis and Caulobacter, we also optimized the esp sequence for Caulobacter for later synthesis with a higher resulting GC content.

Prepared competent cells of E. coli Top10. (Protocol)

Streaked plates of Caulobacter, Staphylococcus aureus, and S. epidermidis.

Template DNA for PCR prepared from colonies using [http://www.bioventures.com/products.php?cid=10005| GeneReleaser]. (Protocol)

Week 2

• 
• PCR products on DNA gel.
  Lane 1: ladder; Lane 2: rsaA from
  liquid culture Caulobacter; Lane 3:
  rsaA from plate culture Caulobacter;
  Lane 4: esp from S. epidermidis.

Performed PCR on esp and rsaA genes (protocol) and ran results on gel (protocol). Results (shown to the right) showed successful amplification.

Performed a transformation experiment (protocol) to test the efficiency of our competent cells make in Week 1. Results were successful with about a 0.7 x 10^7 CFU/μg of pUC19.

Purified PCR product (protocol) and digested esp with EcoRI, SpeI, and PstI; rsaA with EcoRI, XbaI, and PstI; and pSB1C3 with EcoRI and PstI. We then ligated esp, rsaA, and esp and rsaA to pSB1C3 and transformed into E. coli Top10. (Protocol)

Inoculated liquid cultures with colonies from transformations.

Performed digestion of results from transformation. Results shown to the right.

• 
• Gel results for transformation of ligations.
  Lane 1: ladder; Lane 2: digested plasmid
  with rsaA; Lane 3: digested plasmid with
  rsaA and esp; Lanes 4-8: digested
  plasmids from various colonies with esp.