Thoughts

__TO DO__: QPCR:
 * change regression formula for unknowns in 12C qPCR plate ran last week
 * apply new CFUs to graph against plate counts

DNA EXTRACTIONS:
 * List approx how many DNA extractions needed for duration of experiment
 * Make own reagents an option, but may need to optimize DNA extractions = time
 * For eight flasks (2 media, triplicate, 1 control) with 10 time points = 80 DNA extractions for 1 temp/1 pCO2
 * For 3 pCO2 levels at 2 temps: ~500 samples to process (DNA only)
 * Order reagents/kits

PRIMERS:
 * Roberts lab has some primers I am interested in
 * Test these primers to see if these can work for gene expression analysis
 * Work on more primer set development
 * test VtpR primers to see if they work on RE22

pH SPECT:
 * turn in grant app
 * check with CF about sample retrieval - line ok?
 * ORDER!

COMMITTEE MEETING
 * Proposal
 * Presentation

STATS:
 * growth curves
 * qPCR vs Plate Count

8/4/10 - The Plan - LARVAL DISEASE CHALLENGES Get Vt samples from OA experiments set up this week.

Need to know: Inoculation amount - aim for 10^4 CFU/ml to start. The bacteria will continue to grow overnight (~18hrs) before changing water Incubation time - overnight? ~18 hrs Temp - 18C pCO2 - ambient air and 900 ppm Replicates - 4 control pCO2 with larvae, 4 high pCO2 with larvae, 2 controls at each pCO2 __without__ larvae

Samples: 2 - 50 ml conical vials of SW from each tank each day One for DNA and one for RNA extractions Run all the primers for expression data: chiA - need more of these primers vtpA vtpR

Duration of experiment: 3 days?

How many samples do we need for statistical significance?


 * __Larval Challenges:__**
 * Need to figure out:**
 * Dosage for the larval challenges - from Meg's trial my guess is 10^5**
 * Sampling for Vt - when and how much?**


 * __Start CO2 trials with designer gas:__**
 * Two temps 18 and 25C at two pCO2 levels of 840 and 2000 pCO2**
 * I should do some statistical analysis prior to these experiments testing the difference in baseline growth between the 18 and 25C curves. I don't think they are statistically different.**
 * If this is true, maybe it would be more informative to compare 25 and 12C at the different pCO2 levels**


 * __TO DO__:**
 * Bring the tanks of CO2 (840 and 2000) upstairs - 1 in 240 and 1 in 236**
 * Chain them to the walls for support**
 * Set up tubing and regulators for both incubators**
 * Perform the experiments 2 at a time with one temp (18 or 25C) at both 840 and 2000 pCO2**
 * This way we can stay with the sampling schedules used in the baseline trials without coming in every two hours. Instead one time period would be used for 2 pCO2 levels every 5-6 hours depending on temp used.**


 * Figure out what time points we will determine CFUs**


 * __QUESTIONS__:**
 * what about 380 pCO2? Is that scubbed/preindustrial? or just air?**
 * are we ordering this?**


 * Meg's LD50 experiment:**
 * Week of 5/3:**
 * Wed: start with keeping larvae alive until Friday and make equipment for water changes and food**
 * - make pipette tips with 20um screen and rubberbands**
 * - count T. iso and calculate 250cells/ml for feeding**


 * Calculation:**
 * Count** **T. iso** **with hematocytometer to get concentration per mL of your algae culture. Feeding dose is 250 cells/ml to feed one larvae.**


 * If 4 ml is the total volume of seawater containing 40 larvae (1:10 ratio) then we need 10,000cells (250 cells/ml * 40) of T. iso to feed these larvae.**


 * Note: 10,000 cells/0.5 ml = 20,000 cells/ml**


 * If the original concentration was 1 x 10 ^ 6 of T. iso...**


 * //x//(1 x 10 ^ 6) = 0.5ml (20,000cells/ml)**
 * x = 10 ^ 3/ 10 ^ 6 = 1/10^3 = 0.01ml or 10 ul + 490 ul of FSW we need to add to our 4 ml of larvae well.**


 * __V1C1 = V2C2__**
 * __V1 = total volume in rxn__**
 * __C1 = desired concentration__**
 * __V2 = volume to add to stock to dilute into desired concentration__**
 * __C2 = stock concentration__**


 * __Planning for CO2 trials and Vt growth...__**
 * __Potential dates: May 24-27th__**
 * ==**Sampling Times (25ºC and 18C together)**== ||
 * **25** ||  ||   ||   ||   || **18** ||   ||   ||
 * **SAMPLES** || **Sample Time** || **Hours of Growth** ||  ||   || **SAMPLES** || **Scheduled Sample Time** || **Hours of Growth** ||
 * T0 || 6:00 AM || 0 ||  ||   || Time 0 || 6:00 AM || 0 ||
 * T1 || 10:00 AM || 4 ||  ||   || Time 1 || 12:00 PM || 6 ||
 * T2 || 2:00 PM || 8 ||  ||   || Time 2 || 6:00 PM || 12 ||
 * T3 || 6:00 PM || 12 ||  ||   || Time 3 || 12:00 AM || 18 ||
 * T4 || 10:00 PM || 16 ||  ||   || Time 4 || 6:00 AM || 24 ||
 * T5 || 6:00 AM || 24 ||  ||   || Time 5 || 12:00 PM || 30 ||
 * T6 || 10:00 AM || 28 ||  ||   || Time 6 || 6:00 PM || 36 ||
 * T7 || 2:00 PM || 32 ||  ||   || Time 7 || 12:00 AM || 42 ||
 * T8 || 6:00 PM || 36 ||  ||   || Time 8 || 6:00 AM || 48 ||
 * T9 || 10:00 PM || 40 ||  ||   || Time 9 || 12:00 PM || 54 ||
 * T10 || 2:00 AM || 44 ||  ||   || Time 10 || 6:00 PM || 60 ||
 * T11 || 6:00 AM || 48 ||  ||   || Time 11 || 12:00 AM || 66 ||
 * T12 || 3:00 PM || 56 ||  ||   || Time 12 || 6:00 AM || 72 ||
 * T13 || 6:00 AM || 72 ||  ||   ||   ||   ||   ||

__Divided by 24 hr periods = 7 sampling each day__ __Break up by 7 sampling events and have 2 people on for 7 samples, then switch out.__
 * **TIME** || **SAMPLES NEEDED** ||
 * 6:00 AM || 18 and 25 ||
 * 10:00 AM || 25 ||
 * 12:00 PM || 18 ||
 * 2:00 PM || 25 ||
 * 6:00 PM || 18 and 25 ||
 * 10:00 PM || 25 ||
 * 12:00 AM || 18 ||
 * 6:00 AM || 18 and 25 ||
 * 10:00 AM || 25 ||
 * 12:00 PM || 18 ||
 * 2:00 PM || 25 ||
 * 6:00 PM || 18 and 25 ||
 * 10:00 PM || 25 ||
 * 12:00 AM || 18 ||
 * 2:00 AM || 25 ||
 * 6:00 AM || 18 and 25 ||
 * 12:00 PM || 18 ||
 * 3:00 PM || 25 ||
 * 6:00 PM || 18 ||
 * 12:00 AM || 18 ||
 * 6:00 AM || 18 and 25 ||

__Need preliminary trials for 18 and 25C Growth. Maybe take samples every 8 - 10 hrs and see where growth falls when adding CO2.__

FOR EXPERIMENT: __2 negative controls for each:__ __1 flask seawater with air only with Vt__ __1 flask seawater with pCO2 uninoculated__ __1 flask tryptone and seawater with air only with Vt__ __1 flask tryptone and seawater with pCO2 uninoculated__

__3 Experimentals for each media type:__ __3 flasks seawater and Vt with pCO2__ __3 flasks tryptone and seawater and Vt with pCO2__

__Where are we setting this up??__ __If lab:__ __need room for 2 more flasks__ __we are doubling up on these experiments so we need at least 2 big incubators__ __we need more stir plates (ie, another whole set up) in order to accomplish 2 temps at a time__ __need a space and holder for tank__

__If wet lab:__ __we need to reconfigure space__ __should we bring the incubator(s) down?__ __space issues and possible sampling issues since we need to bring all that crap up and down the stairs__

__TO DO:__ __Make lots of plates__ __Make more broth media__ __Order enough syringes and pipettes to handle both temps at once__ __Do we need more 1 L flasks?__ __Need more sponges__ __1ml serological pipettes__ __Mock set up and gathering up of supplies__

__Start molecular work on VptA.__

__**Meeting Minutes for 1-14-10: [|Vt Meeting Minutes]**__ __**Agenda:**__

__For a later discussion…__ //Vt with Cg at different pH and temps –Design//__:__ __3 pCO2 levels: 380, 840, and ~2000 ppm__ __Two temps: 18 and 25C (no 12C?)__

Problems__:__
 * Using seawater for these trials at diff pH and temp
 * how long will Vt stay viable in straight seawater?
 * Sampling from seawater. Need RNA and DNA extraction method for filtered seawater.(Abundance and Gene Expression samples needed)
 * How much Vt do we need to inoculate with?
 * Experimental design discussion with whole group including Cg people
 * Experimental design discussion with whole group including Cg people

Thinking about 16S (what are the drawbacks of using this?), peptidoglycan (OprL), Sec A, filA (sigma 28), LPS core oligosaccharide kdtA or rfaK, Ribonuclease E (RNase E)
 * Need normalizing gene for Vt.**

Lipid A region of LPS RpoS RseA (SR lab) tdh - parahaemolyticus homologous gene
 * Genes of interest for pH trials and pathogenicity**:


 * Need clarification on these things:**
 * 1. Sampling**
 * RNA sampling in seawater other than spinning into pellets
 * How? Is there a kit for filtering and extracting prokaryote RNA?

Vt Growth Curves__:__
 * 2. Experimental Design of Trials**
 * Media - If we will be using seawater in all the trials, should we perform growth trials at different temps in seawater or use media. If media, again, what media should we use? Russ suggested using tryptone/NaCl media with less tryptone to match environmental levels?
 * One curve in sea water and one in media? Plus plate counts Seawater will be difficult. Need much bigger volumes for seawater.
 * Temp ranges: 10, 18 and 25 C (How will we achieve the 10C?)
 * All temp in duplicate
 * OD readings
 * having problems reading OD after 10^-6 - they all have the same readings in further diluted cultures.

pH growth trial:
 * Sterile sampling - use the glass pipettes? Flame sterilize?
 * Air input - is the Designer gas sterile or do we need to rig some 0.22 micron filter tips on there?
 * pH measurements - how are we doing this? is there a way to double check measurements? Should we order another pH probe like the one in the lab?
 * Media. How/What/Need to sterilize - contamination issues/pH control was problematic
 * used 0.22 um filter sterilized T1N2 and it was contaminated the next morning
 * autoclaving the same media turned it cloudy
 * adjusted pH with sodium bicarbonate to ~7.8 (trying to reach 8), next morning the control media was at pH ~8.6! Untouched media with T1N2 was at 7.6.
 * Should we still stick with T1N2?
 * We will use straight sea water for the Cg + Vt trials. Should we just stick to seawater??

Vt with Cg at different pH and temps__:
 * Using seawater for these trials at diff pH and temp
 * how long will Vt stay viable in straight seawater? Do we need to worry about that? Is that long enough to get the info we need?
 * Sampling from seawater. Need RNA and DNA extraction method for filtered seawater.(Abundance and Gene Expression samples needed)
 * How much Vt do we need to inoculate with?
 * Experimental design discussion with whole group including Cg people


 * 3. Molecular Design**:
 * Normalizing gene
 * GOI - environmental response and parthenogenecity of host (2 genes?)
 * Use Lee primers and start cloning
 * Develop standard curve for genomic qPCR
 * Optimize
 * Develop other genes: primers, probes
 * Are there other gene discovery methods I should look at - microarray? protein differences?