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From 2011.igem.org

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 GeneReleaser. (Protocol) Week 2

DNA gel from 20110606 Figure 1: 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. Plasmid result gel for 20110610 Figure 2: 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.

Performed PCR on esp and rsaA genes (protocol) and ran results on gel (protocol). Results showed successful amplification (figure 1).

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 a MiniPrep on the results from transformation, digested the plasmid and ran the results on a gel to check the success of our transformations (figure 2).

Transformed BBa_K081005, a BioBrick part containing a gene for constitutive promotor and RBS, into E. coli Top10. Week 3

Digested Esp plasmid and rsaA PCR product. Ligated Esp plasmid with rsaA and transformed into E. coli Top10.