Team:HiZJU-China/Result
Construction of plasmids
We use the amoA gene and HAO gene from Nitrosomonas europaea ATCC 19718 to enable our chassis bacteria to degrade 17α-ethynylestradiol (EE2).
Fig.1 Gene circuit of amoA,HAO
We expressed the proteins together with T7 promoter and lac operator on the pETDuet-SoGO plasmid backbone
Fig.2 pETDuet-amoA-HAO Map plasmid map
Proof of expression
The constructed plasmid was transformed into E. coli BL21(NB1). Positive colonies that were selected by ampicillin preliminarily (Fig. 3) and then by colony PCR, restriction enzyme digestion (Fig. 4&5) and sequencing were cultivated to express target protein.
Fig. 3 Positive colonies on ampicillin medium
Fig. 4 The agarose gel electrophoresis of enzyme-digested pET-amoA (Nco I & Hind III).
Fig. 5 The agarose gel electrophoresis of enzyme-digested pET-amoA-HAO (Bgl II & Xho I).
The supernatant was extracted from the NB1 culture after ultrasonication. Cells from both samples, original BL21 strain and NB1 strain, can be suspended in SDS sample loading buffer, and ran on an SDS-PAGE gel to verify protein expression (i.e.,a prominent band for amoA protein at about 32.1 kDa while band for HAO protein at about 64.3 kDa) . However, we found the target bands are ambiguous to distinguish on the polyacrylamide gel after staining.(Fig. 6)
Fig.6 (failed)SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining.
Therefore, we optimized the IPTG induction condition and decided to induce cells with 1.0 mM IPTG for 22h at 28℃. Finally, we get the SDS-PAGE reslut of amoA and HAO.(Fig. 7)
Fig. 7 SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining.
Additionally, we assumed that amoA protein, which belongs to membrane proteins, may stay in the precipitate instead of supernatant. So we immersed precipitate in 6 M urea solution in order to solve target protein in supernatant. But it had little impact.(Fig. 8)
Fig. 8 SDS-PAGE analysis of processed precipitate.
After verifying that the two proteins can be expressed through SDS-PAGE, we used ELISA to verify the effect of amoA and HAO on the degradation ability of ammonia by chassis bacteria, meanwhile, we used HPLC-hv to check on the degradation of EE2.
As the operation of HPLC is time-consuming and demanding, we start with ELISA. After being cultured in LB medium containing ampicillin for a certain time, ammonia was partly degraded by NB1. Ammonia nitrogen reacts with Nessler's reagent to form a yellow brown complex which has absorption peak at 420nm. And the content of ammonia in the medium was determined by ELISA at OD420.
Fig 9 the change of concentration of ammonia with time.
Fig 10 the change of content of nitrite ion with time.
Fig 11 the ability to degrade urea.
As illustrated in the bar chart, our engineered bacteria have a conspicuous degradation effect on organic amine like urea，the existence can be reckoned on as the outer nutrient bacteria BL21 gain energy from decomposing organic amine. More over, when the organic amine is transformed into ammonium, to some extent it will sequentially oxidized into NO and released from the liquid phase. This process isn't so fiercely as AOB, which rely on the energy created in ammonia metabolism process.More over, it can be analyzed that there is actually negligable accumulation of NO2- whereas the EE2 degradation will suffer less turbulence, in that way we archive our preset goal of effectively and harmlessly dispose EE2 contaminated water.
Fig 12 the standard curve of EE2.
Fig. 13 Relationship between the peak area of EE2 and culture time.
The content of EE2 in the medium was determined by HPLC. The amount of EE2 was represented by peak area. We found that compared with the blank control, the transformed bacteria could degrade EE2 (Fig. 11).
Fig.1 Gene circuit of amoA,HAO
Fig.2 pETDuet-amoA-HAO Map plasmid map
Fig. 3 Positive colonies on ampicillin medium
Fig. 4 The agarose gel electrophoresis of enzyme-digested pET-amoA (Nco I & Hind III).
Fig. 5 The agarose gel electrophoresis of enzyme-digested pET-amoA-HAO (Bgl II & Xho I).
Fig.6 (failed)SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining.
Fig. 7 SDS-PAGE analysis of amoA and HAO by Coomassie Blue staining.
Fig. 8 SDS-PAGE analysis of processed precipitate.
Fig 9 the change of concentration of ammonia with time.
Fig 10 the change of content of nitrite ion with time.
Fig 11 the ability to degrade urea.
Fig 12 the standard curve of EE2.
Fig. 13 Relationship between the peak area of EE2 and culture time.
Construction of plasmid
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.
Fig. 14 Gene circuit of detection system
We chose pGAD-T7 and pGBKT-7 as our plasmid backbones. Both of them has suitable restriction sites for gene editing.
The two plasmids were combined with GRIP1 gene and ERα-421F gene, which has receive codon optimization.
a
b
Fig.15 ERα-421F gene after codon used ajustment. a, Optimized sequence of ERα-421F gene; b, CAI analysis of ERα-421F gene(CAI: 0.96).
a
b
Fig.16 GRIP1 gene after codon used ajustment. a, Optimized sequence of GRIP1 gene; b, CAI analysis of GRIP1 gene(CAI: 0.98).
The bait plasmids pGBKT7-ERα-LBP and hunting plasmid GADT7-GRIP1D were constructed and introduced into yeasts.
Fig. 17 pGBKT-7-ERα-421F plasmid map
Fig. 18 pGAD-T7-GRIP1 plasmid map
Modification of chasis cells
Our detection system aims to co-relate the content of EE2 with portable readout, such as cell growth and fluorescence intensity.
Therefore, we introduced red fluorescent protein RFP gene and green fluorescent protein GFP into saccharomyces cerevisiae Y2H. We successfully obtained two recombinant fluorescent yeasts-red fluorescent yeast Y2H-RFP and green fluorescent yeast Y2H-GFP. Both of them are regulated by T7 promoter.
Fig.19 The gene was successfully introduced into the plasmid
Proof of expression
Due to the long and unstable sequence of GRIP1, there would be fracture in the process of enzyme digestion and recombination. At the initial stage of the experiment, the full-length positive clone of the gene could not be obtained. (Therefore, the detection stage was carried out later than the degradation stage.) Accordingly, we introduced DNA fragments into yeasts and reconnected them through yeast culturing. Additionally, the integral plasmid structure was sequenced by using pGAD-7 standard primer sequence T7.
Fig.20 yeast Y2H-RFP cultured on different medium shows different color. a, positive control. b, negative control.
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. For yeast Y2H-RFP, the reporter gene RFP mCherry was activated and expressed red fluorescent protein.
Fig. 14 Gene circuit of detection system
b
Fig.15 ERα-421F gene after codon used ajustment. a, Optimized sequence of ERα-421F gene; b, CAI analysis of ERα-421F gene(CAI: 0.96).
b
Fig.16 GRIP1 gene after codon used ajustment. a, Optimized sequence of GRIP1 gene; b, CAI analysis of GRIP1 gene(CAI: 0.98).
Fig. 17 pGBKT-7-ERα-421F plasmid map
Fig. 18 pGAD-T7-GRIP1 plasmid map
Fig.19 The gene was successfully introduced into the plasmid
Fig.20 yeast Y2H-RFP cultured on different medium shows different color. a, positive control. b, negative control.
