Introduction

The final BeeT product will be applied using sugar water which is transported into the hive by worker bees. This means that our chassis needs to be able to survive in a high sucrose, low (non-carbonic) nutrient environment. In order to test this we looked into whether or not any bacteria would survive such an environment over various time steps. The result of this was that our chassis bacteria, Escherichia coli , was able to grow to at least an entire day in the sugar water. This result shows us that the bees will have at least 24 hours to pick up some of our BeeT. Assuming of course the addition of the various sub systems we have in mind doesn't affect the survival time.

Background

The modeling results from the metabolic modeling project only showed what would happen in the time period of 90 seconds to 90 minutes after being put in the sugar water. Because of this, more evidence was needed to see what would happen to the E. coli in the sugar water, for the time scales we were interested in. We know the presence of the high amount of sugar water leads to osmotic pressure. This is because the sucrose molecules from the medium are not able to travel across the cell-membrane, but they still pull the water molecules present inside the cell towards them. The effects of the osmotic pressure on the bacterium were not clear outside of the 90 minute range determined by the metabolic model. However, as determined by the BeeHave model, we require that the BeeT survives for days in the sugar water. In this project we aimed to experimentally determine how long BeeT can survive in osmotically unfavourable conditions.

Materials and Methods

The method used to test the survival of E. coli in sugar water experiment is a simple growth experiment. First the bacteria grow overnight to saturate an LB solution. Then a small amount of this is pipetted into various concentrations of sterile sugar water made of high amounts of sucrose and sterilized tap water. After this they are left there for various time steps and then put into sterile LB medium and left to grow overnight.

Materials used:

Pure sucrose
Tap water
(Multi-)Pipettes
E. coli K12 MG1655 strain, upon which the harvard strain and the iJO1366 model are based.
96 wells plate (well volume ~200 μL)
Plate-reader

protocols applied:

Day before, inoculate LB with E. coli then let that solution grow overnight at optimal conditions (12 hours). This is our saturated LB solution.
Under sterile conditions:
- Put concentrations of 0, 100, 200 and 400 grams sucrose, per Liter of sterilized tap water, plate 1, row 1 and 3. With three replicates each.
- Put LB in all wells of plate 2, close and keep sterile.
- Put bacteria in plate 1 row 1 (sugar water)
- After time steps of 30, 50, 90, 120 minutes inoculate LB wells of plate 2 with 5 μL from row 1 of plate 1. At room temperature with a closed lid./ul>
- For exp 1 a plate reader was used to compare overnight growth curves in the LB. For the other experiments a visual confirmation was enough.
Plate set ups (tables):

Results

[pictures of plates + picture of plate reader results??, showing growth curves overnight] Exp1: failure, did not work sterile (put 96 wells plate on shaker, bad idea) Repeat Exp1: success, growth visible after up to 2 hours in sugar water. See table 1 and figure 1. Exp2: success, growth shown after 6 hours in sugar water. See table 2 and figure 2. Exp3: success, bacteria from exp2 survived overnight. See figure 3.

Table 1. Results of experiment number one, all combinations were done with 3 replicates, which all showed the same result. All rows were interspersed with rows of sterile LB and the final row was inoculated with sterilized sugar water as negative controls.

Sucrose Concentration (g/L)	0	100	200	400
min
30	Growth	Growth	Growth	Growth
50	Growth	Growth	Growth	Growth
90	Growth	Growth	Growth	Growth
120	Growth	Growth	Growth	Growth

Table 2. Results of experiment two, all combinations were done with 4 replicates, which all showed the same result. All rows were interspersed with rows of sterile LB and the final row was inoculated with sterilized sugar water as negative controls.

Sucrose Concentration (g/L)	625	625	312.5	312.5
hour
1	Growth	Growth	Growth	Growth
2	Growth	Growth	Growth	Growth
3	Growth	Growth	Growth	Growth
4	Growth	Growth	Growth	Growth
5	Growth	Growth	Growth	Growth
6	Growth	Growth	Growth	Growth

Discussion

Clearly shows sugar water is not as lethal as first hypothesized, results fit with the model in that survival is indeed possible. (Model just shows the effects that water efflux can have on the bacteria, for the range between 90 seconds and 90 minutes). Recommendation: In order to measure a more quantitative effect of the sugar water, the bacteria could have been put on LB plates instead of wells, and measuring the Colony Forming Units after an overnight growth step.

References