Our ABS system consists of three subsystems, pollution gas indicator system , pollution gas metabolism system, co-culture system. Next we will show the success and failure of the experiment on the three subsystems .
P ollution gas indicator system
In this subsystem, we designed two sets of schemes to indicate two different common indoor pollution gases, HCHO and H 2 S.
- HCHO
Figure 1 ： Colony PCR of recombinant E. coli strain 1aI for Formaldehyde indicating . P.S.：Parallel sample； N.C.：Negative control ； P.C.: Positive control
We added formaldehyde solution to E. coli strain 1aI cultured overnight, continued to culture for 1 hour, and observed under a fluorescence microscope, successfully found that E. coli emitted obvious green fluorescence.
Figure 2 ： E. coli strains 1aI under fluorescence microscope with same field. Left: under exciting light; Right: under white light.
Under excitation light irradiation, E. coli with different concentrations of formaldehyde also showed different degrees of green fluorescence
Figure 3 ： Under the induction of different concentrations of formaldehyde, E. coli emitted different degrees of green fluorescence
We then take more accurate measurements. Different concentrations of formaldehyde were added to strain 1aI, and the changes of fluorescence intensity and OD600 over time were detected under microplate reader.
Figure 4 : The change of fluorescence intensity induced by different concentrations of formaldehyde
Strain 1aI successfully displayed different fluorescence signals for different concentrations of formaldehyde, indicating that the pathway we designed worked successfully .
We normalized the fluorescence intensity by dividing it by OD600 (figure 5).
Figure 5 : N ormalized fluorescence intensity induced by different concentrations of formaldehyde.
The results indicate that under the induction of formaldehyde, the fluorescence intensity of E. coli strain 1aI increased with time and gradually reached saturation. At the same time, the fluorescence intensity increased with the increase of concentration in the range of 0-250μM. After the concentration is greater than or equal to 500μM, the cell no longer grows, the fluorescence no longer increases, and E. coli strain 1aI may not be able to tolerate formaldehyde above this concentration.
- H 2 S
We transformed the designed and synthesized plasmid pET28a-FrmR-sfGFP into E. coli B21 (DE3) and verified the transformation by colony PCR.
Figure 6 : Colony PCR of recombinant E. coli for H 2 S indicating . P.S.：Parallel sample； N.C.：Negative control ； P.C.: Positive control
Unfortunately, the hydrogen sulfide indicating system we designed did not work as we expected. After we added sodium sulfide, the E. coli didn't turn red as expected.
After talking with the team members and our Instructor, we suspect that the reason may be that the expression of the SQR gene is too low to activate the downstream pathway.
To test this hypothesis, we cultured H2S indicated E. coli strain M with another E. coli strain SS(with SQR gene) overnight after adding sodium sulphide. After that, the bacterial solution was made into a temporary slide and observed under a fluorescence microscope, and it was successfully found that some E. coli cells emitted red fluorescence(figure 7). And it was often broken E. coli cell that glowed intensely red under the fluorescence microscope, which confirmed our conjecture. However, due to time constraints, we have not been able to further improve this part of the system.
Figure 7 : E. coli strains M and SS under fluorescence microscope with same field. Left: under exciting light; Right: under white light.
pollution gas metabolism system
In this subsystem, we also designed two sets of schemes to metabolize two different common indoor pollution gases, HCHO and H 2 S.
- HCHO
We configured a series of HCHO solutions with concentration gradient and tested them with detection reagents according to certain methods. The standard curve obtained is ideal. It can be considered that our detection method can accurately reflect the relative content of sulfide in the solution within this concentration range (figure 8) . Note: Limit the fitting curve to pass through the zero point, otherwise the error near the zero point will be too large.
Figure 8 : Standard curve of HCHO
At first, we chose fdh gene from Methylorubrum extorquens and lbfdh (BBa_K2936004) gene from Lactobacillus buchneri to metabolize HCHO. But the results were not good(figure 9).
Figure 9 : Concentration of HCHO in liquid environment with different bacteria.(2b: E. coli BL21(DE3) with pET28a-FrmR- fdh - lbfdh; BL21 ： wild-type E. coli BL21(DE3) ； Control： Blank)
The metabolic capacity of E. coli with the gene related to formaldehyde metabolism was lower than that of wild-type E. coli. By detecting the pH value of the medium, we found that after overnight culture, the pH of the medium of strain 2b decreased significantly (ph≈6). Therefore, we speculated that the reason for the slow metabolism was that the oxidation rate of formic acid was slower than that of formaldehyde, resulting in the accumulation of formic acid, which reduced the pH of the system and the activity of E. coli. Based on this, we made a second try.
A t second time, We chose CboFDH to replace lbfdh.
Figure 10 : Concentration of HCHO in liquid environment with different bacteria.(Cb: E. coli BL21(DE3) with pET28a-FrmR- fdh - CboFDH; BL21 ： wild-type E. coli BL21(DE3) ； Control： Blank)
As is shown in figure 9 , we achieved a satisfactory result this time. Cb has stronger formaldehyde metabolism capacity than wild-type E. coli BL21(DE3). Cb removed 93.0% of formaldehyde within 30min, while wild-type E. coli BL21(DE3) without the corresponding gene only removed 56.7% of formaldehyde. We succeeded in increasing the ability of E. coli to metabolize formaldehyde.
Note: Before testing the formaldehyde metabolism capacity of engineered Escherichia coli, it is best to use LB medium containing some formaldehyde for a few days. This would allow the modified strain to adapt to the formaldehyde environment and increase the number of plasmids containing formaldehyde metabolism genes .
- H 2 S
We did not succeed in this part of the experiment. After successfully transforming the plasmid, E. coli did not show significant metabolic capacity for S 2- . The reason is unknown.
co-culture system
In this system, we introduced Synechococcus elongatus PCC7942 to provide energy and carbon source for E . coli , and introduced SacC gene into E . coli to enhance its ability to metabolize sucrose .
- c oli with SacC
We configured a series of sucrose solutions with concentration gradient and tested them with detection reagents according to certain methods. The standard curve obtained is ideal. It can be considered that our detection method can accurately reflect the relative content of sulfide in the solution within this concentration range.
Figure 11 : Standard curve of sucrose
Figure 12 : Concentration of sucrose in liquid environment with different bacteria
The concentration of sucrose in culture medium of E. coli with SacC gene decreased obviously, indicating that SacC gene successfully enhanced the ability of E. coli to absorb and utilize sucrose.
However, the sucrose concentration in the culture medium of wild-type E. coli increased, which was unreasonable, because E. coli itself did not produce sucrose. We speculated that E. coli produced some metabolites with similar structure to sucrose in the metabolic process, which affected the measurement of sucrose concentration by the kit.
After the sucrose metabolized by E. coli was homogenized, the data of the two experiments were in good agreement(figure 13).
Figure 13 : Sucrose consumption of E. coli BL21 with SacC
S . elongatus PCC7942 with CscB
S. elongatus PCC7942 Slow growth rate (figuer 14) and Nitratireductor sp. contamination events in the process have prevented us from completing the original plan. We completed the conversion of the cscB only two days before freeze day, so there was no time to test the ability of S. elongatus PCC7942 to produce sucrose .
Figure 14 : Growth curve of S. elongatus PCC7942