Team:HiZJU-China/Engineering
This year HiZJU-China have tackled one or more of our project’s problems and use synthetic biology tools to generate desired results.
Combined the teammates’ ideas with advice through Human Practice, we designed three systems for our project, namely the degradation, detection and kill switch system. we found two key enzyme amoA and hao enzyme have the ability to degrade EE2. With the purpose of pursuing better detection methods, we designed yeast two-hybrid technique to detect EE2.
We have done a lot of amazing work to realize the goals we set. The proof on the success of our project is placed here. We hope our experience will serve as a helper for future iGEM teams.
A. Degradation System
1. amoA gene and Hao gene from Nitrosomonas europaea ATCC 19718
The amoA gene encodes the AMO enzyme which produces electrons by ammoxidation reaction. The Hao gene encodes the Hao enzyme. Electrons produced by AMO enzyme are combined with oxygen on Hao enzyme to produce H2O2, which provides oxidant for EE2 degradation.
1-1. Agarose Gel Electrophoresis
Result: Two target gene, amoA gene and Hao gene are designed successfully as the gragh shown below.
Fig. 1 DNA gel electrophoresis of restriction digest product of amoA gene( Nco I & Hind III)
Fig. 2 DNA gel electrophoresis of restriction digest product of Hao gene( Nco I & Hind III)
1-2. SDS-PAGE
Result: amoA gene and Hao gene are expressed successfully, which means that the related gene worked well. We can found amoA and HAO protein both in supernatant and precipitate after cell breakage.
Fig. 3 SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining(supernatant)
Fig. 4 SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining (precipitate)
1-3. Enzyme Activity
Method: amoA and Hao enzymes were used to react with ammonia. We use urea as a nitrogen source. Started testing the rest of the urea and the The products nitrate nitrogen that formed.(OD630 and OD420 respectively). Record the OD420 and OD630 value in real time.
Result: We can see the OD630 of samples decrease very quickly, indicating the degradation of ammonia.
Fig. 5 Enzyme activity of amoA and Hao enzyme(react with urea)
1-4 Ability of engineered bacteria to degrade EE2
Method: Our engineered bacteria were used to react with EE2 and nitrogen. We use a test medium to culture the bacteria. In the test medium, we add urea as the nitrogen source facilitating the cometabolic degradation of EE2. We used HPLC-hv to detect the concentration of EE2 after reaction.
Result: We can found the concentration of EE2 decrease evidently after a certain time, indicating the success of the degradation of EE2. (organic amine will slowly transformed into ammonium.)
Fig. 6 The standard curve of EE2
Fig. 7 The ability of engineered bacteria to degrade EE2
B. Detection System
Our detection system based on yeast two-hybrid technique which required two expression plasmids. The bait plasmid PGBKT7-ERα-LBD carries optimized human estrogen receptor ERα-421F while the hunting plasmid PGADT7-GRIP1 carries GRIP1 gene. The GRIP1 gene can express glutamate receptor interacting protein and be widely used for yeast two-hybid assay.
2-1. Modification of chasis cells
Result: we successfully obtained two recombinant fluorescent yeasts-red Y2H-RFP and green fluorescent yeast Y2H-GFP regulated by T7 promoter.
2-2. ERα-421F gene and GRIP1 gene
Result: Two target gene, amoA gene and Hao gene are designed successfully as the gragh shown below.
Fig. 8 ERα-421F gene and GRIP1 gene
2-3. Protein Expression
Method: When the culture was grown to the logarithmic growth stage, add 5μL EE2 and DMSO with different gradients to 995μL yeasts solution to form exposure culture medium.
Result: For yeast Y2H-RFP, the reporter gene RFP mCherry was activated and expressed red fluorescent protein, indicating the engineered can detect the EE2.
Fig. 9 yeast Y2H-RFP cultured on different medium shows different color. a, positive control. b, negative control.
For the whole result, please visit the result.
Fig. 1 DNA gel electrophoresis of restriction digest product of amoA gene( Nco I & Hind III)
Fig. 2 DNA gel electrophoresis of restriction digest product of Hao gene( Nco I & Hind III)
Fig. 3 SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining(supernatant)
Fig. 4 SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining (precipitate)
Fig. 5 Enzyme activity of amoA and Hao enzyme(react with urea)
Fig. 6 The standard curve of EE2
Fig. 7 The ability of engineered bacteria to degrade EE2
Fig. 8 ERα-421F gene and GRIP1 gene
Fig. 9 yeast Y2H-RFP cultured on different medium shows different color. a, positive control. b, negative control.
