Part:BBa_K1298002:Experience

iGEM CoBRA 2014

• DNA Optimization

The DNA was optimized with Bio Basic Inc.

The original sequence contained illegal cut sites (PstI; ctgca/g)

atggcgggga atgtcgggaa aatgagctcg attttccttg ggacgacgtt agcaatcttc actgccgtgg cgatgatcat gagtttgcct tctgtgtcct cagaggactg cggacagcaa gcaggcggag cgctttgccc cggagggttg tgctgcagca aatggggatg gtgtggcaac acgcaggccc attgtgggca ggattgccaa agccaatgcg gagggggagg ttcgactccc acacccacca cacctacacc gacccccacc actcccaccc ccagcggaca gggagttgca tccatcatga ccgaagatct tttcaatcaa ctgttgaagt acaaagacga cagcagatgc aaggccaatg gattctactc atacgccgcc ttcattgcgg ctgccagtgc tttctccggc ttcggcaccg ccggtgatct cactactaac aaaagagagc tcgctgcttt cttggcccaa acgtcccacg aaaccaccgg cgggtggcag tcggcgccag atggtccgta tgcgtggggt tactgcttta aagaggagca agatcctgtc agcgatttct gccaggcatc ctcccaatgg ccctgcgcat ctggaaagag atactacgga cgaggaccta ttcaaatatc atggaactac aactatgggc aggccgggag tgcactccaa ttcgacggca taaacaaccc ggacattgtt gctagcgatc ccacggtctc ttttaagacg gcggtgtggt tctggatgac cgcccagtct ccaaagcctt cctgccacga cgtgatgacg gggacatgga gtccatccgg cagcgacagc gccgctggca gagcggcggg atatggattg gtgaccaata tcatcaacgg tgggctggag tgcgggaaag gcagcaatgt taagcaggat gaccgcatcg gcttctacaa gcgatactgc gacattcttg gcgtcagcta tggatccaac atcgactgca acagtcagac gccctacgga ggctaa

1. TTGTAG

• Growing

There are no notes to make on the growing techniques or results.

• Miniprep

First to Third attempt: DNA was not staying inside of the wells, but we did not understand why.

Fourth attempt: Determined that our digested DNA was ‘floating’ out of the wells (and not sinking to the bottom) because we were not dry spinning it enough in this step. The ethanol was still with the DNA after dry spinning, even though it should have been all gone. We concluded that the two dry spins that we were doing was not enough to get rid of the ethanol.

Fifth attempt: Dry spun the tubes four times instead of two. This worked and our digested DNA no longer floated out of the wells while pipetting the DNA inside of the wells.

• Restriction Digest & Gel Electrophoresis

Figure 1: Gel Results of Digestion with Restriction Enzymes on J04450 Plasmids Containing Three Different Chitinase Protein Codes using a 1 Kb Invitrogen Ladder (June 14th 2014)

#1: Track It 1 Kb Invitrogen DNA Ladder (the ladder can be found here: http://www.bio.davidson.edu/molecular/Protocols/gels2002/1kbladder.pdf)

#2 - #3: These lanes contain digest results for a different chitinase (See BBa_K1298000 for more details)

#4: This is the PgeChia 1-2 protein code (BBa_K1298002) inside a pSB1C3 backbone. It was cut with the enzymes EcoRI and PstI, causing the coding region to be removed from the backbone. This leaves two parts, the chitinase code (1026 bp) and the backbone (2070 bp), and there would only be two lines showing up on the gel.

#5: This is the PgeChia 1-2 protein code (BBa_K1298002) inside the pSB1C3 backbone. It was cut with only one enzyme (EcoRI), causing the plasmid to be linearized. There is only one part, meaning only one line would show up on the gel.

#6 - #7: These lanes contain digest results for a different chitinase (See BBa_K1298001 for more details)

• Ligation

Figure 2: Initial Positive Results of Ligation

The right-handed plate with red colonies: Red colonies do not have the desired chitinase sequence.

• Transformation

This was performed using DH5alpha competent cells, not NEB Top 10-beta cells.

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