Part:BBa_K4205001:Experience
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Aiming at eliminating heavy metal ions such as Hg2+ and Pb2+ in the intestine of autistic children. We choose Escherichia coli Nissle 1917 as the chassis and pET-28a(+) as the vector. The gene circuit begins with two sensors which can be induced by Hg2+ and Pb2+ respectively and follows the Lpp-OmpA-MT gene, which encodes the protein that can anchor to the bacterial outer membrane and chelate the heavy metal ions. To verify the inducible expression of our functional genes Lpp-OmpA-MT, we induced the bacteria at different concentrations of lead ion salt solution. After lysing the bacteria and performing the centrifugation, we took the supernatant or precipitation at the first or third hour. Then we ran the SDS-PAGE to check MT protein level for all samples.
The pre-stained protein ladder is from 10 to 180 kDa. We induced the protein expression at different concentrations of lead ion salt solution (112 mg/L, 61mol/L, and 30.5 mol/L) and used double distilled water as the negative control. As shown in the figure below, the bands we obtained were nearly 40 kDa, which was almost twice the molecular weight we expected. After careful consultation with our PI and searching literature, we found it has been reported that MT easily forms a dimer in vivo. Hence, we believed this indicated that Lpp-OmpA-MT proteins would combine and form a dimer in the bacteria, which confirmed the expression of our target protein. Then, using ImageJ, we quantitatively analyzed the band brightness and obtained the following results:
- ImageJ analysis: measure the brightness of corresponding lanes, minus the background brightness, and compare it with the marker to get the protein expression of each lane
As shown in the figure above, the protein expression was greater in the precipitation than in the supernatant and was greater after three hours than after one, which was consistent with our expectations. To determine whether our engineering bacteria can grow in the presence of heavy metals, we measured the growth curve. We use MATLAB to fit the data and obtain the figure and formulas shown below.
We used untransformed Escherichia coli Nissle 1917 (wild type) as the control. The concentration of Pb(NO3)2 solution is 112 mg/L. As shown in the figure above, the growth of our engineered bacteria will not be affected by heavy metals. Also, at the logarithmic phase, when the bacteria are most sensitive to changes in the environment, our engineering bacteria grew faster than the wild type, which indirectly proved that our engineering bacteria have the ability to eliminate heavy metals.
As to prove that our engineering bacteria can eliminate heavy metals directly, we put the engineering bacteria into the metal salt solution Pb(NO3)2 and cultured them for 2 hours. Then, we took the supernatant and measured the concentration of the metal salt solution using atomic absorption photometer.
We used untransformed Escherichia coli Nissle 1917 (wild type) as the negative control. As shown in the figure above, the transformed bacteria eliminated more heavy metals than the wild type, and the difference was significant(P<0.05). This indicated that our engineered bacteria were able to eliminate heavy metals. In the future, we will adjust the reaction time with the metal salt solution to further optimize the experiment condition.