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Once we decided on our final bioreactor design, the experimentation fell into two categories, parameter testing and testing out our final complete design.
Parameter Testing
In parameter testing experiments, our goal was to optimize each parameter in order to maximize the amount of horizontal gene transfer (HGT) occurring in our bioreactor. We studied which media and agar to use for our liquid and solid phase, the relationship between bacterial density and HGT, and how agitation of the bioreactor, or lack thereof, impacts HGT levels.
HGT Calculation Method
We used the same HGT calculation method for all of the experiments involving parameter testing. In each experiment, we used two strains of Acinetobactor baylyi, one that has a resistance gene for spectinomycin and another that has a resistance gene for kanamycin (both strains have an additional resistance gene for chloramphenicol to curb contamination). When the two strains are combined and allowed to sit together for a given amount of time, HGT can occur between the strains. After the allotted amount of for the experiment is up, a sample from the experiment is serially diluted and placed on 3 separate agar plates, one with spectinomycin, one with kanamycin, and one with both antibiotics. The agar plates are then left to grow on the agar plate for 24 hours, after which the colonies on each plate are counted.
We know that all of the colonies that survived on the plate with both antibiotics must have antibiotic resistance genes for both antibiotics, meaning that HGT had occurred in the bacteria that those colonies formed from. The counts from each of the plate are used to calculate the HGT level with the following equation.
s = number of colonies on the spectinomycin plate
k = number of colonies on the kanamycin plate
b = number of colonies on the plate with both antibiotics
T = total number of colonies = s + k + b
Media and Agar Composition
Acinetobactor baylyi is typically grown on Luria-Bertaini (LB) agar and in LB broth. We hypothesized that the bacteria may experience higher levels of HGT when growing in a more nutrient-dense environment, which Terrific Broth (TB) could provide. So, we compared the HGT rates of the bacteria in shaking test tubes with LB broth and TB, and the HGT rates on LB and TB agar plates. All of the conditions were placed in a 30 degree incubator for 24 hours, after which we calculated HGT in each of the conditions.
HGT levels in the two broths were similar, with a slightly higher HGT rate in the LB broth.
HGT rates were considerably higher on the LB agar plate than on the TB agar plate.
HGT vs Bacterial Density
Our original assumption was that HGT would be highest at a maximal bacterial density. We tested this assumption by comparing HGT at a variety of bacterial densities. Test tubes with various bacterial densities were allowed shake in an incubator for one hour, after which the HGT levels in each test tube were calculated. The various bacterial densities were made diluting a saturated batch of Acinetobactor baylyi grown in LB at 30 degrees.
Evidently, this experiment did not uphold our assumption. Instead, it appears that HGT levels hit a peak around the 0.083x dilution, and decrease as the bacterial density gets further away from the 0.083x dilution.
Stationary vs Shaking
We hypothesized that some movement of the bacteria would increase HGT as each bacteria would have the opportunity to be in contact with more bacteria. In order to test this hypothesis, we set up two flasks with both strains of bacteria, one of which was placed in a shaking incubator, and the other was placed in a stationary incubator. Both were kept in the incubator for 24 hours.
From the graph, we can see that there was a higher HGT rate in the shaking flask than in the stationary flask. Further experiments will have to be done to determine which agitation method results in the highest level of HGT (ie. stirring or shaking). Future experiments should also be done to determine the ideal agitation speed.
Growth Rate Experiments
Growth rate experiments were performed to ensure that the two strains can grow together in the same bioreactor. Our bioreactor will only be successful if the two strains have a similar growth rate, meaning that neither of the strains will be flushed out when left to grow in the bioreactor over time.
We found that the strain with the kanamycin resistance gene had a growth rate of 0.0306 sec-1 and the strain with the spectinomycin resistance gene had a growth rate of 0.0293 sec-1. Since the two growth rates are very close, we concluded that these two strains are good candidates for our bioreactor.
For a continuous stirring tank reactor with media flow in and out of the system:
𝞵 = D = F/V
𝞵 = CSGR | D = Dilution Rate |F = Fin = Fout = Flow Rate | V = reactor volume
𝞵KAN/CHLOR=0.0306 | 𝞵SPEC/CHLOR=0.0293
Therefore, taking the slowest CSGR to avoid flushing out
Optimal Flow Rate of the Bead-o-Stat = 0.0293*(volume)
This is the OD measurement from a run of our bioreactor with the Chi.Bio system. The OD measurement represents the bacterial density in the bioreactor.
Written by Sarah Davydov
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