In figure 1, the bands on lane No. 2, 3, 6, 7, 12 represented successful plasmid construction. Comparing to the ladder, our bands have a size of roughly 7.7kb, which is the length that we are looking for.

Figure 2. Gel electrophoresis results for BamHI digested Mer-GlnA-GFP(pDu) plasmid.
In figure 2, the lanes are counted in a right to left, up to down fashion.
Lanes number 4, 13 and 14 show bands at roughly 7.7 kb according to our ladder separation.
We also expect the length of our digested product to be at this length, so we can conclude that the plasmid construct is successful.

Figure 3: Gel Electrophoresis result for Mer-GlnA-GFP(pDu) plasmid

The length of Mer-GlnA-GFP(Tn501) plasmid should be 7.7kb. Figure 3 shows that the length of plasmids we constructed are 7.7kb.
We concluded that the Mer-GlnA-GFP(pDu) plasmids were successfully constructed.

The length of our LacI-GlnA-GFP plasmid should be 5.5kb. Figure 4 shows that samples in well 1, 2, 6, 10, 12 have the correct length.
We concluded those LacI-GlnA-GFP plasmids were successfully constructed.

Demonstration:
_____We have successfully constructed four plasmids: 3 Mer-GlnA-GFPs with different binding sites (RBS, Tn501, pDu1358) and 1 LacI-GlnA-GFP.
In the assessment of the growth of E. coli with Mer-GlnA-GFPs, bacteria did not respond to different concentrations of mercury ion as we expected.
We found that the concentrations of mercury ion were too high. However, we did not have enough time to test the growth of our engineered E. coli with lower concentrations of mercury ion.
The control experiment where glutamine was added, the bacteria proliferate normally. This result proved that auxotrophic mechanism of our engineered bacteria worked.
_____We constructed LacI-GlnA-GFP plasmid to test if the ion deficiency auxotrophy system is going to work.
We used the IPTG binding to LacI to simulate the situation of mercury ion binding to Mer gene. The 48hr kinetic result of E. coli containing LacI-GlnA-GFP gene plasmid showed that the growth of the engineered E. coli was affected by concentrations of IPTG.
The concentrations of IPTG we used were 0, 0.025, 0.125, 0.25, 0.5, 0.75, 1, 1.5 mM. The higher the concentration, the faster the bacteria grew (lower Ks constant in Monod model).
When the concentration of IPTG was 0, the bacteria did not grow. The growth curve of E. coli in media without IPTG showed a flat line with no observed growth. During the 48 hours kinetics, both fluorescent intensity and OD600 was measured.
Fluorescent intensity characterized the expression level of glnA, and OD600 indicates bacterial concentration.

Figure 1: The OD600 curves of 48hr kinetic for sample 1 in different IPTG concentrations

Figure 2: The OD600 curves of 48hr kinetic for sample 2 in different IPTG concentrations

Figure 3: The OD600 curves of 48hr kinetic for sample 3 in different IPTG concentrations

Figure 4: The Fluorescence curves of 48hr kinetic for sample 1 in different IPTG concentrations

Figure 5: The Fluorescence curves of 48hr kinetic for sample 2 in different IPTG concentrations

Figure 6: The Fluorescence curves of 48hr kinetic for sample 3 in different IPTG concentrations

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Team:Minnesota/Results

Team:Minnesota

#Minnesota 2018

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