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From 2011.igem.org

Notebook

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• 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.
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• 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.
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• Figure 3: lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA that had been digested 20110606; lane 5: PCR product of plasmid containing esp; lanes 6-8: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA PCR product from 20110605. Only lane 6 shows successful ligation of esp and rsaA.
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• Figure 4: lane 1: ladder; lane 2: PrsaA PCR product; lane 3: Pxyl PCR product

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

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

Inoculated cultures of transformed BBa K081005. We performed a Miniprep on the overnight cultures to obtain plasmid containing BBa K081005.

PCR amplified prsaA, a constitutive promotor in Caulobacter and pxyl, an inducible promotor in Caulobacter.

Digested plasmid containing esp and PCR product of rsaA. Ligated esp plasmid with rsaA and transformed into E. coli Top10.

Prepared plasmid samples containing rsaA and esp for sequencing.

Picked colonies on transformation