Part:BBa_K3076100
Coding sequence of metallothioneins (MT) gene from Corynebacterium glutamicum
Metallothioneins (MT) are proteins made of 61-68 amino acids with a small molecular weight. It is found in almost every known organism, from bacteria to humans. Due to this ubiquity, it minimizes the gene toxicity effect when we choose it as the ectopic expression target.
MT contains many cysteine groups which were reported to be responsible for chelating a wide range of metal ions such as cadmium, lead, copper, and mercury, etc. Nevertheless, MT from different species showed different affinity towards different metal ions.
Literature reported that MT protein from Corynebacterium glutamicum (CgMT) shows strong binding affinity towards divalent cations, such as Zn2+ and Pb2+. [1] Since our project used Cu2+ as a model for the metal pollutants, we decided to explore the plausibility of using CgMT to increase the metal accumulation ability of E. coli.
Furthermore, Corynebacterium glutamicum belongs to the same order as Mycobacterium tuberculosis. Previous iGEM team Oxford 2016 demonstrated that MT from M. tuberculosis (BBa_K1980002) chelates copper ions in cells effectively, thus we expected CgMT also chelates copper (II) efficiently and it will increase the accumulation of copper ions inside E. coli expressing CgMT.
This part was synthesized into pET151/TOPO vector for the ectopic expression in E. coli BL21. The results showed that the CgMT transformant absorbed significantly more copper ions (~15%) in the culture medium when compared with non-induced control and empty vector control.
Assay 1: Test for copper absorption ability on E.coli transformed with CgMT
Abstract of experiment
We utilized pET-151/TOPO vector to drive the expression of CgMT coding sequence under T7 promoter inside BL21(DE) E. coli strain. Then, the bacteria transformants will be tested inside copper (II) sulphate added culture medium to measure their copper absorption efficiency along time.
Protocol:
1. Prepare overnight culture of MT BL21(DE) E. coli transformants and pET28a(+) BL21(DE) E. coli empty vector controls (Incubated at 37oC, 80 rpm)
2. Transfer overnight culture to freshly prepared Luria broth medium with antibiotics (Ampicillin for MT transformants and Kanamycin for empty vector control) in a ratio of 1:50. Then incubate at 37oC, 80 rpm for 2 hours.
3. Add IPTG into the designated IPTG+ culture and Empty vector control culture to a final concentration of 0.1 mM. Then incubate at 37oC, 80 rpm for 4 hours.
4. Adjust the concentration of bacterial culture using LB broth and the absorbance of each culture (IPTG+, IPTG- and Empty vector) is normalized to 1.5.
5. Add 0.5 mL of 100 mg/L copper (II) sulphate solution to each test tube for assay.
6. Add 4.5 mL of corresponding bacterial culture to each test tube.
7. The experimental groups are designed according to the table below:
Table 1: Time of incubation in 0, 1, 4, 16 hours with engineered bacteria
| Sample | Time of incubation (Hours) | |||
|---|---|---|---|---|
| 0 h | 1 h | 4 h | 16 h | |
| (IPTG+) 4.5 mL CgMT Transformant with IPTG + 0.5 mL 100 mg/L CuSO4 MT1 gene | 0 h | 1 h | 4 h | 16 h |
| (IPTG-) 4.5 mL CgMT Transformant + 0.5 mL 100 mg/L CuSO4 MT1 gene | 0 h | 1 h | 4 h | 16 h |
| (Empty) 4.5 mL Empty vector Transformant with IPTG + 0.5 mL 100 mg/L CuSO4 MT1 gene | 0 h | 1 h | 4 h | 16 h |
| (Blank) LB medium MT1 gene | 0 h | 1 h | 4 h | 16 h |
8. When the designated time point is reached, 3 mL of each bacterial culture from the group is centrifuged with 6,000 rpm, 1 minute.
9. 2.5 mL of the supernatant is transferred to the cuvette and added with 175 uL of API copper testing reagent. Pipette up and down to make sure the reagent is completely reacted.
10. Measure the absorbance of the reaction mixture by colourimeter with 440 nm. Repeat the measurements twice to ensure no further development of colour.
11. Record the absorbance and convert it to concentration value by the standard curve.
Graph 1: Percentage decrease of copper concentration along time
Graph 1: The percentage decrease of copper concentration in the culture medium In the graph, the percentage change of copper(II) ion concentration along time is shown. The copper added media were incubated with IPTG+ (IPTG induced CgMT expression E. coli), IPTG- (CgMT expression E. coli without IPTG added) and Empty (Empty vector control E. coli). IPTG+ showed ~30% decrease, IPTG- showed ~18% decrease and empty vector control showed ~14% decrease.
Results
The result shows that IPTG+ (blue) had a significant increase in copper removal ability when compared with IPTG- control (red) and Empty control (green). The purple curve shows the blank control in which no bacteria were inoculated in the copper added medium.
It indicates that CgMT expression contributes to the copper absorption ability of the E. coli strains (BL21). Although IPTG- also showed a statistical difference when compared with empty vector control, we believe it was due to the small sample size or leaky expression of the T7 promoter. Further investigation is required. This assay was repeated twice and the error bars represent standard deviation which indicates the distribution of data range.
Future work
Since our assay method is relatively insensitive when compared with using advanced techniques such as mass spectrometry, we are going to collaborate with university labs to seek a more sensitive way of measurement and thus we can generate a model with higher precision.
Secondly, we aim to create an E. coli absorbent of heavy metals so we are going to combine CgMT with metal exporter knockout modification and further increase the absorption ability of E. coli.
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