RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec’d. RNA was stored @ -80C in “Shellfish RNA Box #4“.
Results:
All gill RNA looks nearly perfect (based on 260/280 and 260/230 values). Muscle RNA is only OK (based on 260/280 and 260/230 values).
Attempt to obtain full genomic sequence for C.gigas COX. PCR set up/cycling params/etc are here. Primer set combinations(master mixes) are as follows:
MM01 – Cg_COX_5’UTR_3_F (SR ID: 1150) + Cg_COX_1009_R (SR ID: 1147) Band size w/o intron = ~1000bp
MM02 – “” + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~1540bp
MM03 – “” + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~2135bp
MM04 – Cg_COX_982_F (SR ID: 1151) + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~550bp
MM05 – “” + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~1130bp
MM06 – Cg_COX_1519_F (SR ID: 1146) + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~620bp
Results:
Bioline Hyperladder I used for marker. Gel is loaded with template samples at the far left of each master mix group with two no template controls (NTC) in the remaining two wells of each master mix group. All NTCs on the gel are clean.
All bands surrounded by a green box were excised from the gel.
MM01, MM02 and MM03 – The smallest expected band (i.e. no intron present) would have been 1000bp in MM01. Instead, we see faint banding of multiple sizes less than 1000bp in both MM01 and MM02. MM03 fails to produce any bands. This potentially suggests a couple of things. Firstly, the multiple banding produced in MM01 and MM02 suggests that the PCR conditions lead to some non-specific priming and should be optimized. Secondly, the fact that no bands were produced that are equal to or larger than the “no intron size” suggests that intron(s) may exist in the 5′ region of the COX gene and are large enough that the polymerase had insufficient time/ability to amplify.
MM04 – Three distinct bands were produced: 2000bp, 1500bp and 550bp. The size of band that would have been produced had an intron NOT been present would have been ~550bp. A band of this size was produced in this PCR reaction. However, two additional bands were produced. The presence of these two larger bands lends additional evidence for the existence of multiple isoforms of COX (which is also supported by the fact that multiple isoforms of COX are known to exist in most other species). The 2000bp band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.
MM05 – A distinct band of ~5000bp was produced. This is significantly larger than the “no intron size” of ~1130bp, suggesting the presence of an intron. This band was excised, but not purified due to the low concentration of DNA in the gel. The gel slice was stored @ -20C until this PCR reaction could be repeated to accumulate sufficient product for sequencing.
MM06 – A distinct band of ~2200bp was produced. This is significantly larger than the “no intron size” of ~620bp, suggesting the presence of an intron. The band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.
The PCR reactions reveal the presence of intron(s) in the COX gene we’re investigating as well as providing evidence for the existence of multiple isoforms in C.gigas. Since the PCR products that have been excised for sequencing are so large, additional primers will need to be designed closer to the introns in order to generate smaller products that can be fully sequenced. Additionally, all reactions using the primer designed to anneal in the 5’UTR of COX (SR ID: 1150) failed to produce useful results. This is either due to poor performance of the primer under these reaction conditions or due to the presence of a large intron in the 5′ region of the gene. Additional reverse primers will be designed that anneal closer to the 5′ portion of the COX gene in hopes of characterizing the 5′ genomic sequence better.
After speaking with Steven today about the potential existence/”discovery” of multiple isoforms, he decided to map the newly-released C.gigas 454 NGS data to the existing COX coding sequence in GenBank (FJ375303). The alignment is shown below.
The two 454 reads that map closest to the 5′ end of the COX coding sequence match up nearly perfectly, with periodic SNPs. The remaining 454 reads that map to the COX coding sequence are very different and provide very good evidence of a previously unidentified isoform of COX in C.gigas. Primers will be designed from both the existing COX sequence in GenBank (FJ375303) and the other potential isoform. These primers will likely be used in both qPCR and for sequencing purposes, in order to be able to distinguish and characterize both isoforms. Additionally, BLASTing will be performed with the sequences from both isoforms to evaluate how they match up with existing COX isoforms in other species.
All dilutions were performed with 1x LB+ 1%NaCl. 100uL were plated of all dilutions (see below) on 1xLB+1%NaCL plates. Plates were incubated O/N @ 37C. Colonies will be counted tomorrow to determine CFU for each sample.
Plated 100uL of:
V.vulnificus, t=0, 1:1,000,000 and 1:10,000,000
V.vulnificus H2O sample, t=1 & 3, 1:10,000 and 1:1,000,000
V.tubiashii, t=0, 1:1,000,000
Control H2O sample, t=1 & 3, Undiluted
Samples tubes containing bacteria and dilutions were stored @ 4C.
UPDATE 20110112:
Colony counts and calculations
V.vulnificus 0hr = Both dilutions produced a total lawn of bacteria. Uncountable. Will plate higher dilutions, but this will now only be a rough estimate due to the time that has passed.
—UPDATE—
New serial dilutions (90uL plated) of V.vulnificus 0hr were performed, down to 10^12. The only countable plate was the 10^12 dilution.
V.vulnificus 0hr = 10^12 dilution x 410 CFU = 4.1×10^14 CFU/90uL = 4.56×10^12 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 3.64×10^19 CFU total in oyster tank/8L = 4.56×10^18 CFU/L
V.vulnificus 1hr = 1:1,000,000 dilution x 48 CFU = 4.8×10^7 CFU/100uL = 4.8×10^5 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 3.84×10^12 CFU total in oyster tank/8L = 4.8×10^11 CFU/L
V.vulnificus 3hr = 1:1,000,000 dilution x 23 CFU = 2.3×10^7 CFU/100uL = 2.3×10^5 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 1.84×10^12 CFU total in oyster tank/8L = 2.3×10^11 CFU/L
Control H2O (no significant growth occurred O/N, just tiny colonies; continued incubation to allow colonies to increase in size for easier counting)
Control H2O 1hr = Undiluted 146 CFU/100uL = 1.46 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 1.168×10^7 CFU total in oyster tank/8L = 1.46×10^6 CFU/L
Control H2O 3hr = Undiluted 106 CFU/100uL = 1.06 CFU/uL x 8×10^6uL (8L H2O in oyster tank) = 8.48×10^6 CFU total in oyster tank/8L = 1.06×10^5 CFU/L
V.tubiashii 0hr = 1:1,000,000 dilution x 31 CFU = 3.1×10^7 CFU/100uL = 3.1×10^5 CFU/uL x 5×10^4 (50mL total volume of bacteria) = 1.55×10^10 CFU total V.tubiashii
400mL O/N culture (1x LB+1% NaCl, 37C, 150RPM, 1L flask) of V.vulnificus (STRAIN??) and V.tubiashii (Strain: RE22) were pelleted (4300RPM, 25C, Sorvall ST-H750 rotor). Supe was removed and pellets were each resuspended in 50mL sea water. 1mL was taken from each to use for dilutions to determine colony forming units (CFU).
Two containers were set up with each containing 16 C.gigas, and air stone and 8L of sea water. The entire 50mL of V.vulnificus was added to one of the containers. 8 oysters were sampled (gill and mantle tissue) from each container at 1hr and 3hrs after the addition of V.vulnificus culture and immediately frozen on dry ice. Samples were stored @ -80C in the “Gigas Vibrio Exposure 1,3hrs 1/11/11″ box. Additionally, 1mL samples of the water were taken at each time to determine CFU in the water.
In addition to the samples taken above, the following tissues were taken from 5 control oysters at the 3hr time point and treated/stored in the same fashion as the others, specifically for assessment of cyclooxygenase tissue distribution analysis: muscle, digestive gland/gonad (difficult to differentiate)
All oysters were measured. Morphometric data is here.
Retrieved the following SOLiD libraries from the CEG and stored in -80C in the “NGS Libraries” Box.
CC SOLiD Library (20010408 13.8ng/uL)
Herring 2L HKOD09 SOLiD Library (20091209 76.1ng/uL)
Herring 3L HSITK09 SOLiD Library (20091209 88.5ng/uL)
Herring 4L HTOG09 SOLiD Library (20091209 20.1ng/uL)
Herring 6L HPWS09 SOLiD Library (20091209 51.4ng/uL)
HPWS09 SOLiD Library (20010408 9.29ng/uL)
PQ SOLid Library (20100408 20.77ng/uL)
Sisco SOLiD Library (20100408 42.29ng/uL)
CE SOLiD Library (20100408 23.01ng/uL)
Used COX primers (SR IDs 1060, 1061) and cDNA from 20080327, which consisted of 7 control gigas gill and 7 vibrio-exposed (24hrs) gigas gill samples, labeled as C# and VE#, respectively. The experiment was a 24hr. exposure live Vibrio vulnificus, parahaemolyticus Cf = 2.055×10^11 (6.85×10^7 Vibrio cells/oyster).
Note: Used a free sample of 2x Brilliant III Ultra Fast SYBR Green QPCR Master Mix (Stratagene) for this qPCR. Mixed components and set up cycling params according to the manufacturer’s recommendation for the BioRad CFX96.
Master mix calcs are here. Plate layout, cycling params, etc. can be see in the qPCR Report (see Results).
Results:
PCR Miner analysis is here. There appears to be an increase in COX expression in samples exposed to Vibrio sp. (see graph below), however, I have not determined if the results are significant.
Used new cyclooxygenase primers (SR IDs 1060, 1061) to see how they performed and to evaluate tissue distribution. Tissue distribution was evaluated using the following cDNAs made on 10/27/10 from Emma:
Gigas Digestive Gland
Gigas Gill
Gigas Mantle
Gigas Muscle
qPCR Master Mix calcs are here. Plate layout, cycling parameters, etc can be found in the qPCR Report (see Results).
Results:
qPCR Report (PDF).
Amplification is present in all four tissue types and the melting curve looks good. So, these primers are good to go. Steven suggests checking to see if we see a change in gene expression from an old experiment of Gigas exposed to high levels of Vibrio tubiashii. Will round up some old cDNA for this.
Reaction calculations are here. Samples were mixed and incubated @ 37C O/N (started at 6PM). Mac will take care of them tomorrow morning.
1st of 2 rounds of digests were performed. Calculations are here. Samples were incubated 37C for 3hrs, heat inactivated @ 80C for 30mins and then stored @ -20C.
Samples were EtOH precipitated, according to protocol. Samples were resuspended in 20uL of Qiagen’s EB and spec’d.
Samples are labeled as Parent (P), #, tissue, enzyme (MspI = M, HpaII = H, Undigested = U)
Results:
Spreadsheet of spec values is here. Overall, poor recoveries from all of the digested samples, but decent recoveries from the undigested samples. The samples were passed to Mac who performed qPCR using two different primer sets. Please see her notebook for the results of the qPCR.
