Team:Aalto-Helsinki/LabResults
Laboratory Results
The constructs
Overview
Our goal was to produce propane from cellulose in E.coli. To achieve this our aim was to build three plasmids and transform them to the same bacteria. Two of the plasmids include the ten genes necessary for propane production in E.coli. The third plasmid contains the three genes encoding the enzymes for cellulose hydrolysis. To ease the bottlenecks caused by two of the enzymes in the propane pathway we built an amphiphilic protein biobrick. To validate our amphiphilic protein we created a new, fusable GFP biobrick. Amphiphilic micelle formation was validated with electron microscopy.
Propane producing plasmid 1: CAR
CAR is the first of our propane producing plasmids. The plasmid was constructed to pSB6C1 backbone with Gibson assembly. Afterwards the CAR insert was transferred to the biobrick backbone pSB1C3 with restriction digestion and ligation.
Achievements:
- Gibson assembly with pSB6A1 backbone successful according to colony-PCR
- Successfully transferred CAR construct to biobrick backbone pSB1C3
Propane producing plasmid 2: AtoB
AtoB is the second of our propane producing plasmids. The plasmid was straight built to the biobrick backbone pSB1C3 with Gibson assembly.
Achievements:
- Gibson assembly with biobrick backbone pSB1C3 successful according to restriction analysis
Amphiphilic protein
Ampihilic protein was created to fuse the two bottleneck enzymes of our propane pathway close to each other.
Achievements:
- Amphiphilic protein biobrick was validated with sequencing
- Amphiphilic protein was proven to form micelles inside the cell by electron microscopy
Fusable GFP
To help testing our amphiphilic protein we built a fusable GFP biobrick. The fusable GFP biobrick has one additional nucleotiode prior to it’s suffix: it can be fused with any aminoacid’s amino terminus without losing it’s reading frame.
Achievements:
- Created a fusable GFP biobrick
- Fusable GFP was validated with sequencing
- Successfully fused the fusable GFP biobrick with ampiphilic protein
Propane production
Because of the time limit, our propane producing E.coli strain wasn't competely constructed. We decided to try small scale and chemostat production with E.coli BL21 (DE3 ΔyjgB ΔyqhD, pET-TPC4 + pCDF-cAD + pACYC-Fdx-Fpr) made by Kallio et al. which differs from our own strain by two enzymes at the beginning of the reaction pathway. Thus, experiments with Kallio's strain largely reveal how our own strain would have behaved. More information available at the Project page.
Small scale production
Before trying to grow new cells in a reactor, the strain had to be tested with vial-scale cultivations. In this way we could gather information about the induction, cell growth rates in TB-media, and some estimates of how much time is needed for propane formation after adding IPTG. At the same time we could practise the usage of gas gromatography/mass spectrometry and make proper standards for propane detection. Basically, terrific broth (TB) containing induced bacteria would be added into 22ml GC-vials. The cells are incubated different lenghts of time and propane accumulates into the head-space of the vial where the injection and analysis would happen. The protocol contains more detailed information for the preparation of samples.
Two samples were incubated for 17 h with 50 RPM shaking in 22 ml vials. Propane concentrations were 111 μg/L and 88 μg/l where the litres were as in the volume of cultivation media. Furthermore, strain's growth rate was significant as the OD600 (1 ml cuvettes) increased from 0.1425 to 0.5212 for 1h 10 min time scale. The rate was assumed to be much lower because TB-media contained four different antibiotics which were needed to keep plasmids inside of bacteria. Details about standards, peaks etc. for GS/MS can be seen in this file. The GC/MS analysis and calculcations were done by the personnel of VTT.
Continuous production
0.5 L chemostat experiment with 44 ml/h flow rate was successful. With 1.0 L/h aeration, the propane content of the reactor's gas phase was determined to be 4.5-22.7 μg/L with GC/MS 145 h after starting the experiment. The results were calculated when 0.1-0.5 μg of propane was gathered from reactor's gas exhaust into a 22ml gas chromatography vial. However, these values were below GC-standard line so the accuracy may have decreased. The GC-samples were also gathered when a third steady state was running which had much lower cell density than second steady state had. Nevertheless, the result proves that propane can be produced industrially with continuous production.
The batch phase took six hours, and soon after starting the process the amount of dissolved oxygen decreased to zero. Therefore, oxygen became the limiting factor of cell growth. Continuous phase was started and the first steady state was reached 39 h after starting to feed fresh media into the reactor. Overall, the steady state lasted for 15 hours. The density of cell population was calculated to be 9.85 g/L at this point, which can be assessed relatively high. Interestingly, our bacteria acted as a facultative anaerobe when changing its oxygen uptake depending on the growth phase.
IPTG induction started with 2mM concentration and the population growth started to react to changed process conditions. New steady state was achieved 40 h later and the population density became 9.28 g/L. Thus, there was a 0.57 g/L difference of the biomass between the normal growth and the growth with propane production. For some reason, the growth started to decrease again and the second steady state didn't continue, when the feed stock was changed to a bigger container containing the same media. Because propane samples weren't gathered from the second steady state, we had to wait until the third steady state had settled. At this point the cell density was 2,625 g/L. Reactor's glucose consentration decreased from original 20 g/L to zero during the exponential grow phase as the cells used it as a carbon source. However, it was hard to recognize whether the limiting factor of the growth and production was glucose or oxygen. During the first steady state, glucose concentration increased when the need for the catabolism of biomass was lower.
Before starting to feed fresh media, the end of batch phase was determined from decreased pH-level when the cells produced acids as a response to nutrient deficiency. TB-media used for cultivation contained phosphates which had buffer capacity but it wasn't enough. pH-control was assembled in order to maintain pH-values above 5.0.
More information about the experiment available at the Process data sheet and the Lab book. Details about standards, peaks etc. for GS/MS can be seen in this file.