Into the Brine!

Hi Friends, I'm working hard to polish up the deliverables (build guide, code base, user manual, etc), but also proving the capabilities of the current prototypes with some cycling campaigns I've been wanting to try for a long time. One key path I wanted to explore with Vibrio natriegens is having them transition from marine conditions into salt-free media. To do this, I started a culture with 50mL of LB30 (Yeast Extract 5g/L, Tryptone 10g/L, and 30g/L NaCl) and wired up a liter of LB0 (same as above but zero salt) to the first input pump. The initial optical density triggers were set to 0.6 and 0.3 respectively (assuming OD940 of 0.5 is mid-log phase). I let the robot cycle this culture for 60hrs, and after six cycles, the data looked like this:

 

Vibrio natriegens cycling with LB0 media from LB30 over 60hrs

We see the initial cycle from a saturated culture which took a bit over 3hrs to reach OD 0.6. This dilution continues, and given the OD940 bounds of 0.6 to 0.3, the cells will be diluted with 14mL worth of LB0 media, mixed in, and removed to maintain a constant 50mL per dilution cycle. Some quick math shows this equal about a 28% reduction in salinity per cycle. If we started with 30g/L NaCl in 50mL, that equates to 1.5 grams of salt in the initial media. Since we're diluting using LB sans salt, this first lowers the salt to 1.08g (21.6g/L). The table below shows the salinity per cycle (assuming perfect dispensations and aspirations per cycle).

Extrapolating these culture parameters to 20 dilution cycles, we can see an asymptote where only after 10 cycles do we get below 1g/L, and a salinity similar to standard LB media (10g/L) is reached by the 4th cycle.

 

 

Each dilution stretched the time between cycles longer and longer, but the last cycle seemed to have been so stressful that the upper OD trigger of 0.6 was unreachable, so I had to pause the program and reset the bounds to 0.3 upper and 0.1 lower. As soon as I started the program with the new parameters, the robot forced a cycle (because culture OD was above 0.5). The next two cycles looked like this:

 

Again the subsequent dilution could not reach the trigger level so I adjusted the OD bounds one last time to 0.2 upper and 0.05 lower. I let the bot cycle over the weekend, and my mom sent me a pic of the robot while I was out of town showing the new bounds allowed for at least 4 more cycles. Looking forward to the data readout on Monday. By now the culture has experienced at least 13 cycles which equals a salinity of around 0.4g/L.

 

Something cool I noticed is that as the salinity decreased, so too did the "thickness" of the data (noise variance), noted by a thicker fuzz of each subsequent growth curve prior to dilution. This could be due to the cells swelling to account for the osmotic shock, increasing their ability to block or occlude the light from the OD940 LED. As the "graininess" of the cell culture increases due to cell size or clumping, a larger variance in the light scattering can be measured. I still need to prove this idea is true, but once validated we have yet another interesting metric to use as a measurement of cell fitness.

 

Another thing I noticed is that I've been cycling the cells in stationary phase instead of exponential. Regardless of the reasoning, the vibrio cells in my system and current media experience an exponential phase of growth between an OD940 of 0.1 to 0.3. Irony, lol.

 

There are benefits to cycling in stationary phase but would like to keep things moving as fast as possible so the lesson learned here is to always characterize your strain with a batch culture to know where the bounds for cycling should be. More data and insights sooooooon :)