Team:BGIC-Global/Demonstrate

DEMONSTRATE

Demonstrate

Description
After many times of verification and repetitive experiments, we successfully proved our engineered system ca work in real life. Volatilization test of formaldehyde gave us clear and standard control group for our bacteria experiments that decomposing speed of bacteria could directly be figured out by comparing the volatilization curves with bacteria and that of the control group. We also successfully proved functions of EGFP, pFrmR, and GFA that when we mix the BL21 strain having pFrmR+EGFP with the strain having pJ23119+GFA, fluorescence activity was largely higher than in the control group which didn’t have any transferred parts.

1. Volatilization of formaldehyde:
Before the tests on engineered strains, we did preliminary experiment of testing volatilization curve of formaldehyde to make a standard volatilization line that can be compared with the lines when bacteria are added. we hoped to find plateau of about 0.6 mg/m^3, which is the maximum formaldehyde concentration around furniture in new houses. Therefore, we diluted formaldehyde solution from its original concentration of 37percent to five gradients: original concentration, diluted 10times, diluted 100 times, and diluted 1000 times, and they were tested volatilization curve separately.

figure1: original concentraion

figure 2: diluted 10 times

figure 3: diluted 100 times

figure 4: dluted 1000 times

As figure 4 shows, when the formaldehyde solution was diluted to 1000 times less than its original concentration, its volatilization curve reached plateau when concentration was around 0.645mg/m^3. We had successfully found the most similar formaldehyde environment with that in new house before we conduct our experiments on bacteria. In our future experiments, volatilization curve with bacteria would be compared with that shown on figure 4, allowing us to directly distinguish working efficiency of our engineered system.

2. Fluorescence of Nluc:
NanoLuc (Nluc) luciferase is a small enzyme (19.1kDa) engineered for optimal performance as a luminescent reporter. The enzyme is about 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase using a novel substrate, furimazine, to produce high intensity, glow-type luminescence. The luminescent reaction is ATP-independent and designed to suppress background luminescence for maximal assay sensitivity. After getting kit provided by Promega, we tested Nluc’s luminescence activity that we transferred the plasmid into E.coli BL21 strain. a common bacterial strain for protein expression. After overnight culture and adding substrate, we were able to witness the luminescence of Nanoluc in contrast of normal bacteria.

As the graph shows, BL21 with Nanoluc transferred was brighter than normal BL21, distinguishable by eyes, proving that Nluc could be expressed in BL21. Then we combined Nluc with Promotor pJ23119 to test it in formaldehyde condition. This test didn’t have useful results which could indicate performance of Nluc. We deduced the possible reasons could be excessively high formaldehyde that can inhibit growth of E.coli and activity of promotor pJ23119.

3. Responsiveness of pFrmR to formaldehyde:
As mentioned in our part session, pFrmR is a formaldehyde inducing enhanced promotor that theoretically can activate expression of GFP (green fluorescence protein) which is on its downstream when formaldehyde is present. In order to test the responsiveness of pFrmR to presence of formaldehyde, we conducted a verification experiment to test pFrmR performance. The results would be indicated by strength of GFP activity, the strength of fluorescence

Summarized graph for all groups of pFrmR and BL21. In solution with 1ul of formaldehyde, luminescence was the most obvious, indicating more active pFrmR.

In solution with no formaldehyde addition, BL21 with EGFP had similar activity of fluorescence with the control group.

In solution with addition of 0.2ul of formaldehyde, BL21 with EGFP had similar activity of fluorescence with the control group, which means in this condition the activity of pFrmR was low.

In solution with addition of 0.5ul of formaldehyde, the difference in fluorescent degree between BL21 with EGFP and the control group was clear, indicating higher activity of pFrmR.

In solution with addition of 1ul of formaldehyde, the difference in fluorescent degree between BL21 with EGFP and the control group was the most obvious, indicating higher activity of pFrmR which successfully activated GFP.

In solution with addition of 1.5ul formaldehyde solution, the difference in fluorescent degree between BL21 with EGFP and the control group was clear, indicating the high activity of pFrmR.

In solution with addition of 1ul of formaldehyde solution, the activity of pFrmR was the highest.

Experiment group and the control group both had five concentration gradients of formaldehyde added separately into tubes which had 1 ml of bacteria solution in each. As the graph shows, BL21 with pFrmR and GFA had strongest fluorescence when added with 1ul of formaldehyde saturated solution, which means pFrmR had strongest activity under this concentration of formaldehyde, and the negative feedback system was reduced to the weakest that the activity of GFP on the downstream expressed more.

We also did test of mixing BL21 had pFrmR+EGFP with BL21 had pJ23119+GFA.

As the graph shows, when the two types of strain are mix evenly together on ratio of 1:1, fluorescence expression was enhanced even more than that of EGFP only. The possible reason could be in the solution of EGFP only strain, although the promotor pFrmR had highest activity with addition of 1ul of formaldehyde, it didn’t mean that bacteria also express high degree of EGFP because due to concentrated formaldehyde condition, most of the bacteria could inhibited or killed. Nevertheless, in the solution with both of the strains existing, GFA can decompose formaldehyde at the very beginning of addition of it that the great condition in the system was better for bacteria’s survival. Therefore, less bacteria will be killed or inhibited and thus higher activity of EGFP will be shown on the picture as the high degree of fluorescence.

4. Activity of GFA:
Description:

It is an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction, which catalyze the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene GFA is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. The glutathione-dependent formaldehyde conversion to formate starts with the adduct formation, formaldehyde reacts with the SH group of glutathione producing S-hydroxymethylglutathione.

We conducted verification experiments to ensure GFA was active in BL21.

Figure 1: BL21 with pFrmR+EGFP only

Figure 2: BL21 with GFA and BL21 with pFrmR+EGFP

Comparing figure1 and figure2, it is clear that figure two indicates higher fluorescence expression, with higher start point and higher plateau period. In fact, The figure shows the highest expression of fluorescence among all groups of experiment we conducted.

The experiment group only had half amount of EGFP bacteria compared with the control group B, but produced higher expression. The only way to explain this result is that GFA played an important role to degrade the formaldehyde, therefore decreased the toxicity of the environment of bacteria and enhance the survival rate. This also indicates that our engineered system works.

Because of some errors appeared in our engineered system before, there was a period of time that we conducted countless times of verification experiments with no expected results coming out. And now we have the expected results and high expression of fluorescence; we will continue our work and will finally fulfill our aim of creating a terminal product which can detect and decompose formaldehyde simultaneously.