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Team:Vanderbilt/Notebook
From 2014.igem.org
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Lab Notebook | ||||||||||
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Spring 2014 
March 27th
March 30th
March 31st
April 1st
April 2nd
April 3rd
 April 4th
April 5th
April 7th
April 24th
April 25th 
April 27th April 29th Summer 2014 May June '''July'''
* Finally reached the point that all terpene genes were consistently amplifying with the synthase gene primers. The genomic DNA sample for sabinene was completely used before this point, although all other of the 8 terpenes showed clear bands.
 * The results of the genomic DNA PCR indicated each gene had a large fraction of introns. None of the genes had a distinct band at exactly the right weight corresponding with what the intron-less cDNA size would be. 
Fall 2014
August
*Moved the lab into its new space before the start of the semester
*Created the plasmid intermediate pVU1400A, which is missing only a single insert to become pVU14004.
September
* Ran RNA extraction on all the plants that were still available. This excluded Myrcene and Linalool (R) since both Perilla frutescens and Mentha aquatica had withered over the summered.
*Repeated RNA extractions on those which showed appreciable concentration on the nanodrop. Eventually all 7 remanining terpenes had plant RNA in appreciable quantities (most between 20-50 ng/ul, with a few less than 10 and a few more than 100 ng/ul).
September 17th
*Digested plasmid intermediate grown in demethylated bacteria with ClaI. Ligated final insert into vector. No transfomants grow after 24 hours.
*Diagnostic digest shows the ClaI enzyme is cutting properly. pUC19 positive control for transformations show that the competent cells are working.
September 18th
*Ran reverse transcription PCR on extracted RNA to isolate synthase cDNA. Humelene and sabinene show clear positive results, santalene shows amplification at smaller region, and cadinene shows no cDNA bands.
September 19th
*Made liquid cultures of K546546 in preparation for mutagenesis. Also made glycerol stock to store at -80.
* Diagnostic digest of ligation of pVU1400A intermediate and the final insert needed to make finished plasmid. Gel clearly shows bands in exactly the correct positions for each of three comparison conditions, proving that the creation of pVU14004 was finally successful.
September 20th
*Miniprep of K546546 liquid cultures (1 ml ). First culture tube concentration of 85.7 ng/ul DNA, second 105.1 ng/ul
*RNA extracted arabadopsis and Picea abies to improve yield and quality. Carene still failed to get an RNA concentration greater than 10 ng/ul, while Arabidopsis produced 107 ng/ul with a good A260/A280 ratio.
September 20th
*Miniprep of K546546 liquid cultures (1 ml). First culture tube concentration of 85.7 ng/ul DNA, second 105.1 ng/ul
September 26th
*RT-PCR done on humelene, linalool (S), sabinene, and zingiberene. Sabinene produces clear bands, while zingiberene shows one faint band at roughly the correct size. Positive controls are also run to confirm that the reverse transcription step is not the cause of any failures to amplify.
October 5th
*RT-PCR done on humelene, sabinene, and santalene. Sabinene again produces clear bands,and santalene does as well although much fainter. Both bands were gel extracted to yield a small (<10 ng/ul) amount of DNA.
*Extracted DNA was ligated into pUC19 and transformed into E. coli.
October 7th
*Site direction mutagenesis kit and specially designed primers were used to mutagenize K546546 at its BglI site, sabinene cDNA at its XbaI and EcoRI sites, and pVU14004 at its EcoRI and XbaI sites.
*Mutagenesis product was transformed into E. coli.
October 9th
*Minipreps were done on 5 ml liquid cultures of mutagenized pVU14004, sabinene, and K546546. 4 liquid cultures were made for each, and both sabinene and pVU14004 were done in duplicate.
*Diagnostic digests were done on miniprepped plasmid to check if the restriction sites were mutagenized. pVU14004 appeared to have lost its XbaI site but not its EcoRI site, sabinene shows a size that suggests it failed to ligate as an insert into pUC19, and K546 shows only a single band at around its starting weight.
October 10th
*All 8 minipreps of sabinene ligated into pUC19 were digested with SpeI and ApaI to check for the synthase insert. Only one, Sab B2, shows a second band at the right size.
October 11th
*Santalene synthase was ligated into pVU14004 and pSB1C3. E. coli was transformed and incubated.
*The sites that failed to show mutagenesis were mutagenized again using and transformed into E. coli. K546546 had its AgeI site mutagenized.
October 12th
*All liquid cultures were miniprepped, producing 8 samples of pVU14004 with confirmed XbaI mutagenesis, 4 pVU14004 with no sites confirmed, 4 sabinene, and 6 samples produced from santalene in pVU14004. None of the plates with santalene in pSB1C3 produced colonies.
*Diagnostic digests were run on all miniprepped plasmid (K546- AgeI, BglI, SphI. Sabinene- EcoRI, BamH1. Santalene in pVU- ApaI, XbaI. pVU- EcoRI, XbaI, BamHI, KpnI). Santalene appeared not to have ligated into pVU14004. Sabinene did not have its EcoRI site removed by mutagenesis. K546546 had at least one cut, but the second sample may have had one site mutagenized.
*Santalene was re-ligated into pSB1C3 and transformed.
October 13th
*Santalene in pSB1C3 was miniprepped to good yield. Each of 4 replicates was digested with SpeI and ApaI to test for ligation. The ApaI enzyme appears not have cut, but the fourth sample showed an uncut plasmid size which corresponded to that of pSB1C with santalene successfully inserted.
October 14th
* Ligated santalene synthase again into pVU14004 and transformed into E. coli
October 15th
*One colony grew and was put in liquid culture.
*Culture miniprepped and digested. Again no gene insertion was detectable.
October 16h
*Transformed pVU14004 into a dam- strain of E. coli to address the methylation sensitivity of ApaI.
*Finished planning and acquiring materials for GC-MS confirmation of terpene presence.
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