Team:DeNovocastrians/Contribution
Bioremediation is a growing and valuable industry which is combating one of the biggest problems faced by humans and the planet – pollution. Since the first reported use of implementing microbes to treat spills and human-generated hydrocarbon in 1975 (Beneficial Stimulation of Bacterial Activity in Groundwater Containing Petroleum Products, by R. L. Raymond and coworkers (1975)), our scientific understanding of molecular genetics and synthetic biology has boomed. These technological advancements have given scientists the remarkable ability to control and manipulate protein expression, and it was these foundations that our project was built upon.
Our core hydrocarbon degrading strain
The benABCD cluster was chosen for cloning (into the workhorse organism Escherichia coli) because it is one of the best characterised aromatic hydrocarbon degradation pathways – targeting the highly toxic pollutant benzene. The cluster was isolated from Rhodoccocus, Acinetobacter and Escherichia species known to degrade benzene and the much-harder to degrade poly-cyclic-aromatic hydrocarbons.
By cloning in the benABCD cluster there is a proof of concept that can be built upon, and future developments can be made to degrade the more complex poly-aromatic compounds.
Our cloning of benE into a
We have also laid the groundwork for developing a benzene biosensor that could be used in bioremediation efforts. The biosensor works by benzene or catechol inducing the binding of transcription factors to a binding region and recruiting RNA Polymerase. The polymerase then transcribes the downstream fluorescent protein gene, which is later translated into the fluorescent protein, giving the biosensor user a visual notification of hydrocarbon presence.
We hope that future iGEM teams will be able to build upon the research we have completed. This could include altering the expression systems of the benzene degradation and importer proteins; which would allow for a bacterial system to be optimised as a more efficient degrader of benzene. This degradation cluster could also be included with other hydrocarbon break-down pathways such as the catechol degradation pathway, and pathways from Rhodococcus that target poly-cyclic-aromatic hydrocarbons, to develop a bacterial system that breaks down multiple environmental hydrocarbon pollutants.
A further change that could be implemented involves containment upon release. We envisage that the engineered bacteria will contain a "kill-switch." The switch would work as follows:
1) Engineered bacteria (containing only hydrocarbon degradation pathways and no other catabolic pathways) are dispersed in an environment polluted with petrochemicals.
2) Once the pollutants have been degraded, the engineered bacteria have consumed their preferred carbon source.
3) The lack of carbon source kills off the population of engineered bacteria due to an inability to create energy for themselves.
Our biosensor construct
The biosensor we created will be useful in the detection of pollutants and identifying environments that require remediation. Further work is required to determine the detection limits and dynamic range of the biosensor, to accurately equate biosensor readings with an actual concentration level of pollutants in the environment. This is necessary to correctly identify the locations that require the most urgent remediation.
The biosensor construct we developed utilises regulatory sequences from Acinetobacter baylyi (ADP1) and the fluorescent proteins fuGFP and mCherry.
Trouble shooting
The biosensor construct was unable to be synthesised in one part. The transcription factors and fluorescent proteins were synthesised, however, the binding and promoter regions couldn't be synthesised. Due to this, binding regions were amplified out of ADP1 genomic DNA by PCR. These regions contained highly repetitive sequences that were A/T rich, which may have caused non-specific binding. The DNA smear that was observed when the PCR products were run on an agarose gel may have been caused by this non-specific binding. These varied PCR products resulted in difficulty joining the construct together via overlap extension PCR.
Consequently, we needed to re-evaluate the suggested annealing temperatures of primers from the DNA synthesis company. This was done by performing a gradient PCR and discovered different annealing temperatures were needed for the primers. Using traditional overlap extension PCR procedures, there was a low rate of success in joining DNA fragments together and we utilised an optimised overlap extension PCR protocol. Using this protocol we had a much higher success rate.
We successfully attached the gene encoding for the mCherry fluorescent protein upstream of the catM binding region. The introduction of this construct into the catM encoding organism ADP1 will inform us if catechol is in solution by giving off mCherry red fluorescence. Utilising the same concept, we have used the benM transcription factor and its binding region, which were found directly upstream of the benABCD cluster. benM ligand is benzene, which when bound to its binding region activates transcription of downstream genes. In this construct green fluorescent protein (GFP) is directly upstream of the benM binding region. Thus, this construct will inform us if benzene is in solution by giving of GFP green fluorescence.
Microbial growth curves & MIC data
Growth curves are used by scientists to study the differential effects of a given media, population genetics, and the effect of stressors on the growth of a microbial population. The growth curves we have constructed (and the methodology behind them) of Rhodococcus strain 9, Acinetobacter baylyi ADP1 and E. coli BW25113 can be used by future iGEM teams to construct their own growth curves if they are looking to utilise similar degradation pathways. Our growth curves can be used as reference points to characterise the standard and post-gene insertion metabolic characteristics of each of the species we have used. The benzoate and catechol MIC's that we determined can also be used as a guide for future iGEM teams; when deciding on how to develop their own analysis of growth characteristics/kinetics. The growth curves and MIC's determined can also be used in the planning of future team's experiments. For example, they may look at the concentrations we have used to assess metabolic characteristics, then set their own concentration ranges accordingly. Interestingly, we also found that our original E. coli lab strain BL-21 grew in benzoate, causing us to select a different strain. The BL-21 strain was able to consume benzoate as a sole carbon source without being recognised as having a benzoate degradation pathway. This problem may be encountered in other lab strains used by future teams. As such, future teams would be careful in strain selection by first running a gene search on their strains to elucidate the presence of any hydrocarbon degradation pathways.
Parts
BBa_K3694027
benE gene obtained from ADP1 genomic DNA - encodes for a transmembrane symporter which enables cells to import benzene/benzoate from their environment.BBa_K3694028
benE gene obtained from synthesized E. fergusonii DNA - encodes for a transmembrane symporter which enables cells to import benzene/benzoate from their environment.BBa_K3694029
benE gene obtained from Rhodococcus strain 9 genomic DNA - encodes for a transmembrane symporter which enables cells to import benzene/benzoate from their environment.BBa_K3694023
The benABCD gene clusters isolated from Rhodococcus strain 9, is the benzene degradation pathway degrading benzene into catechol. The cluster encodes for a benzoate 1,2-dioxygenase (benABC) which converts benzene into Cis-1,2-dihydroxycyclohexadiene. This is then converted to catechol by the benD encoded dehydrogenase.BBa_K3694024
The benABCD gene clusters isolated from Acinetobacter baylyi (ADP1), is the benzene degradation pathway degrading benzene into catechol. The cluster encodes for a benzoate 1,2-dioxygenase (benABC) which converts benzene into Cis-1,2-dihydroxycyclohexadiene. This is then converted to catechol by the benD encoded dehydrogenase.BBa_K3694026
Biosensor gene construct that would work in E. coli. The construct contains transcription factor catM and its binding site connected to a downstream fluorescent mCherry gene. The transcription factor, in the presence of its ligand catechol or cis,cis-Muconate, would bind its binding region to recruit RNAP and transcribe the downstream fluorescent protein gene. The construct also contains a benM transcription factor and its binding region which is connected to a downstream green fluorescence gene. In the presence of benzene, the transcription factor binds to the binding region to recruit RNAP and transcribe the downstream fluorescent protein gene.Results
New documentation to an existing Part
We have contributed information to the existing part (BBa_K2228000) characterised by the 2017 Purdue iGEM team. The Purdue team also used synthetic biology to tackle the problem of benzene pollution; specifically they "Designed a benzene-degrading genetic circuit that could be incorporated into a non-pathogenic strain of E. coli, and subsequently introduced to other species within the lung microbiome." The benzene degradation cluster (benABCDE) Purdue 2017 identified was isolated from Pseudomonas putida. We also used this degradation cluster, however after literature review and a NCBI BLASTp search, we found the cluster to also be encoded for in Rhodococcus strain 9, Escherichia fergusonii, and Acinetobacter baylyi (ADP1). Due to logistics and ease of use, we opted to use the species we discovered that encode the benABCDE cluster in our project.