Working on experimental design for Bermuda 2024

[NatalieDreux]

Me and the B2P 23 cohort will be designing a incubation experiment, taking place in Bermuda, as part of the C-CoMP program. The main task for now is to come up with a rough draft of how we want to preform this experiment. Taking into consideration things such as: the water budget, what measurements we are taking, sample types (bio/tech), etc. This will be a deliverable for the 3/8/24 B2P weekly meeting with Vicki, where Liz will be in attendance to answer some questions.

[NatalieDreux]

My main job

Providing information on the metabolomic aspect of the project. i.e. How much water per samples (5mL), storage, etc.

[NatalieDreux]

Reached out to Liz for clarification on metabolite mixes

[NatalieDreux]

Experiment Questions

1. Are we sampling for DNA at just the beginning and the end or are we going to sample for DNA at each timepoint?

Subsequent question: how much will sampling throughout add to the project cost?

2. What is the expected timeline for the incubation? Are we shooting for 24 hours or 48 hours?*

   • longer timeline would allow for more changes to take place within the community which would be good for DNA analysis (not as much change is going to take place on the community level in a 24 vs a 48 hr period, have to give them time to divide/change) Are we aiming to do duplicates or triplicates for the treatments? We have to consider water budget (sounds like we have access to 24L)

3. Are we going to want biological replicates or technical replicates

   Procedure Notes/Questions

4. Water samples provided from Liz, sampling from surface level. We are electing to use whole water and not filter our larger organisms Usure of sample quantity/size

   Genomics: Samples should be ~500mL filtered down onto Mill Metabolomics: Samples should be at least 5mL, meaning ~15-20mL per time point accounting for triplicates and excess:

   if technical replicates the sample would be aliquoted into 3 separate 5mL samples (similar to the Pro11 MIT experiment). If biological replicates, 5mL samples from each ‘community’ [ i.e. each incubation (control, Pro, Micro.) ]

5. Incubation period of 24(?)hrs with timepoints of T0,Tx,Ty,T24

Could do T0, T6, T12, T24 T0, T12, T18, T24 T0, T8, T16, T24

• Max: i think that we should focus more on the front end of the incubation period rather than the end

3. Max’s Timepoint Proposals for Different Time Frames (we don’t need to use these, they’re just suggestions.)

   24: 0, 6, 12, 24

36 0, 12, 24, 36

48 0, 12, 24, 48

• (only reason i wouldn’t go further than 12hrs for the second point given the large time frame is because I really do think the front end of the experiment is the most important but given a time frame like 48 hrs i think getting points 12 and 24 would allow for a relatively early timepoint (12) that would provide initial(ish) uptakes of the metabolites and then 24 and 48 would allow for a decent enough time for divisions to take place for community structure change to occur. Same line of thinking applies to the reasoning behind the 36 hour points.

4. Are metabolites and genomics being measured at each of these timepoints or is it just metabolites and genomics only at T0 and TFinal?

   More so thinking about the genomics

5. We ideally will have a control and two other sets for our two organisms metabolite mixes. If we did two treatment groups and a control, 4 timepoints, and duplicates, this would likely use all 24L of our water allotment.

   Example: at T0 samples would be taken as follows ~

   • Control A
   • Control B
   • Treatment 1A
   • Treatment 1B
   • Treatment 2A
   • Treatment 2B

6. Using metabolite mix from Prochlorococcus and Micromonas

   • The metabolite mix is developed around the phytoplankton exometabolite experiment in which each of these organisms were grown from an axenic culture and metabolites measured during exponential growth phase.
   • The metabolites present are the excreted metabolites from our source organisms.

     So the implication being that whatever is growing and consuming the metabolites during the incubation period are favorable towards the excretions of the organisms we took the metabolite mix from. This could reveal some interesting dynamics between the phytoplankton and microbes present if there are differential observations between samples.

7. Genomic investigation to be done in Dyhrman Lab

   • Who/where is metabolite extraction/identification done?

     This is most likely happen at WHOI as sending samples with chemicals is a hassle from Bermuda and there will likely be no time to do the PPL SPE needed for analysis to have ‘dry’ samples to send back

     ID will also be at WHOI using LC-MS, Skyline, etc.