Team:MSP-Maastricht/Contribution
Contribution
Parts
Mbb1 - BBa_K3947000 & Mbb4 - BBa_K3947002
Gene sequences that were identified in the seaweed Asparagopsis taxiformis by (Thapa et al., 2020). It is an enzyme from the family of bromide peroxidases and catalyzes the following reaction:
HBr + H2O2 = HOBr + H2O
The enzyme uses vanadium as a cofactor in the catalysis of the bromoform. The active site features a vanadium oxide center attached to the protein via one histidine side chain. The enzymes catalyze the oxidation of bromide by hydrogen peroxide. Bromonium cation (Br+) that is generated attacks hydrocarbons (represented as R). They are only specific to the bromide and are unable to oxidize chloride at all.
R-H + Br- + H2O2 = R-Br + H2O2 + OH-
Mbb2 - BBa_K3947001
A family of enzymes that are generators of reactive oxygen species, which are called nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOXs) for short. They are membrane bound enzymes and catalyze the transfer of the electrons from the electron donor, NAD(P)H, via flavin and heme cofactors to molecular oxygen generating a reactive Oxygen Species – hydrogen peroxide and superoxide anion.
Mbb2 enzyme is distinct from the other NOXs due to the positioning of the membrane-spanning heme-binding/oxygen reducing domain as well as the flavin-binding/NAD(P)H-oxidizing cytoplasmic domain.
CcVHPO1 - BBa_K3947003 & CcVHPO3 - BBa_K3947004
This gene was retrieved from Chondrus Crispus (Thapa et al.,2020). This gene is analogous to the Asparagopsis taxiformis bromoperoxidase. They follow the same principle and mechanism as the A.Taxiformis enzymes. It is an enzyme from the family of bromide peroxidases and catalyzes the following reaction:
HBr + H2O2 = HOBr + H2O
The enzyme uses vanadium as a cofactor in the catalysis of the bromoform. The active site features a vanadium oxide center attached to the protein via one histidine side chain. The enzymes catalyze the oxidation of bromide by hydrogen peroxide. Bromonium cation (Br+) that is generated attacks hydrocarbons (represented as R) They are only specific to the bromide and are unable to oxidize chloride at all.
R-H + Br- + H2O2 = R-Br + H2O2 + OH-
Spacers
Spacer System - BBa_K3947005
We wanted to be able to make our research sustainable and the genes easy to manipulate. Therefore, a spacer-gene-spacer system was created. This would allow our genes not only to be separated from one another but give us the flexibility to produce various combinations. This gives us the possibility and ability to implement kill switches or gates at any location that would aid in regulating the function of the genes themselves.
Composite Parts
Gate Switch - H2O2 Regulation
This device is divided into multiple parts: the device consists of an OxyR (BBa_K1104200) transcription factor that detects H2O2 and will activate transcription. It will activate the AhpC (BBa_K362001) promoter initiating the process of translation. If there is not H2O2 present in the system, then there will not be any production of genes.
OxyR is a transcription factor that is dependent on a Reactive Oxygen Species (ROS), in this case our Mbb2 (BBa_K3947001) will be the producer of this. Once the OxyR senses the present H2O2 (ROS) it will go through a change and create an intramolecular bond. The ahpC promoter on the other hand will start the process of gene translation only when the OxyR is able to bind to it and initiate the process of translation. To Recap if no H2O2 is present in the system then the genes will not be created.
Kill Switch With Hairpin
Following the concept of the Nebraska-Lincoln iGEM team of 2017 we developed a hairpin structure that at high temperatures such as 37C would be linear. In this part of the hairpin structure, we placed a modified RNase E cut site that would be able to create a break and stop the translation of the gene. However, when under low temperature conditions such as ambient room temperature, roughly 27C, the hairpin will be generated and there will be no cut site that the RNase can cut and thus the nucA (BBa_k11159105) will not be generated. The nucA is a deadly degrading endo-exonuclease. It will degrade everything: dsDNA, ssDNA, dsRNA and ssRNA.
Ruminal Simulation
A ruminal simulation was built to test our product. In nature, Methanobrevibacter, which are responsible for methane production in cattle’s rumen, inhabit an anaerobic environment. In order to test our bacteria’s effectiveness, our team wanted to recreate a cow's ruminal compartment, with conditions as real as possible.
For our contribution, we created a series of tutorial videos that explain our process and thinking behind the simulation, and explain how to set up such a simulation, with emphasis on ensuring anaerobic conditions, as well as lab safety.
Such a construction would be essential for the testing stage of any project which needs to do microbiome analysis. Moreover, due to the simplicity of the building process and availability of components, the simulation is highly adaptable for recreation of internal processes of any live animal.
Watch the tutorial of the assembling process here: (Hardware Award Page)
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