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Team:CityColSanFrancisco/Notebook/LabBooks/Colby
From 2008.igem.org
6/25
Lab book created. All future work done by me will be posted on this document.
Following yesterday's plating of S. oneidensis plates were observed to search for growth. No growth was detected. This is rather disturbing as oneidensis is supposed to be our fastest grower. What went wrong? Perhaps our media is bad or maybe our incubator is running too hot/cold.
We will test our media first. New media was made using same procedure as previous batch (the only difference being that 20g of agar was used instead of 15g). Plates were poured using this new batch. We will determine if our LB (from the previous batch) is defective by plating 1 plate from our previous LB, 1 from a plate borrowed from the Btech program, and 1 plate from our new batch of LB. Hopefully there will be some growth by tomorrow on at least one plate.
6/26
No growth in Shewanella plates, again. Thermometer reads 38C, a full 8 degrees hotter than our bacteria prefers. This may be responsible for our fledgling shewanella. Plates were moved to a new incubator, this one set at 29C. The plates will be left in there over the weekend, with Nicola checking on them.
6/29/09
Growth found in several Shewanella plates. Temperature seems to have been a factor. Growth was found in all but the Btech plate. A gram stain was preformed, to determine the identity of the bacterial strain. Results: a gram negative rod shaped microbe. Shewanella matches this, but we are still unsure if Shewanella is really there We will design a primer using a unique sequence, or a sequence of a unique length, known for Shewanella. This will determine our bacteria’s true identity. One such primer possibility is O-succinylbenzoate synthase ATGATCTTAACTTCACTTAGCTTGTATTTATATCGCCTGCCCTTAGACAGCTTTTTGCCTGTGGGCAAACAACGCATTGACCATAGAAAAGGCTTAGTGCTGCAAGCCAAGGCCACTGCT GACGGTGAAGTTTGCGACACCGAAATATCCAATGCTGAAGTGACTGACAGTGAAGTCACTGAGGGTGAAGTGAAGGAGTCCCATGTTGAAATCGCGCCACTCTCAGGGTTCGATATCGAC CAACAGCCATTATCCGGCTTTAGCCGCGAAAGCCTCGATGAAGTACAGCAAGCCTTAACAGTGTTATTACCCAAGCTGCAAAACCAATCTATTGATTGCTTGCTCGAGCAGGCCGAAGCG AGTCCCTATCCATCCATTGCCTTTGGCTTAAGCCTATTGCATGCCAAACTCAGCGGAAAACTCGATGCGGTGCGCCCACTCACCACCGCTGTACCTTTAATTTATCAACCAACTGATGCG CCTAAAGCTGAATTAATAGCAAAAATTGCCAGCCTCAAACCTAGTGTGCGCTCAGTTAAAGTGAAGGTTGCACAAACCTCCATGGAAGATGAGTTGAGTTTGATTTATGGCATCTTAAGC CAAAGGCCCGATCTCAAGCTGCGCTTAGATGCCAATTGCGGATTTAGTCTAGAACAGGCCCTCGACTTTGCCGCCTGCTTGCCACTAGATAGCATTGAATACATTGAAGAACCTTGCCAA CATCCGCAGGATAATCACACTCTGTATCGCGCCATTCCACTGCCCTACGCCTTAGATGAATCCCTCAATGATCCCGATTATCAATTTGTGATGCATGAGGGACTCACGGCGCTCATCATC AAACCCATGCTACTGGGCAGTATCGAAAAACTTCAGCGCCTTATCGATGAAGCCCACAACCATGGCGTGCGCTGTATCTTAAGTTCCAGCCTTGAAAGCAGTTTAGGCATCAATGATTTG GCCCATTTAGCCGCCATACTAACGCCCGATGAAATCCCCGGGCTTGATACCTTAACGGCCTTTAGTCAAGACTTATTAGTCCCATCGGGCAAACTACAATGTCTTACGCTTCAATGCCTG ACACTGAATCAACTCGAACGAGTCGCCAGCACTGTACAGGATTGAT
Sequence (5'->3') Strand on template Length Start Stop Tm GC% Forward primer GGTGAAGTGAAGGAGTCCCA Plus 20 184 203 60.09 55.00% Reverse primer TCGAGTTTTCCGCTGAGTTT Minus 20 425 406 59.99 45.00% Product length 242 Template 4769986 .................... 4769967
Product length: 242 Construction of the primer will continue tomorrow. From S. oneidensis 6/25, streak one plate using (new) LB media, 1 (old) LB media, and inoculate liquid culture. From P. aeruginosa 6/26, streak one plate using 1 (new) LB plate, 1 (old), inoculate liquid culture. Fro mutant S. oneidensis freezer stock , steak on 1 LB plate (new) and 1 LB plate (old). Inoculate palustris in liquid YP culture.
