"As a man evolves, Nature dissolves"
Once destroyed, the beauty of nature cannot be reacquired. Humans are causing life on Earth to vanish. Carelessness leads to catastrophic events. When the industrial discharge is released into the water bodies, the heavy metals present in the effluent cause significant damage to the environment, leading to mass disturbances in the terrestrial and marine ecosystems. Cadmium in specific is found to be mutagenic when exposed for a long period of time.
In this page we have shown the results of our recombinant gene expression and reactor model.
Our aim was to produce a solid biofilm structure that would contribute to the production of our hero enzyme, Acid Phosphatase. E.coli K12 strains possess poor biofilm abilities. With the introduction of csgD and OmpR genes, we enhanced the biofilm strength of E.coli K12. OmpR upregulation followed by csgD activation and csgD protein production induced the levels of curli fibers responsible for biofilm formation.Presence of OmpA linker facilitated the cell surface display of acid phosphatase enzyme.
Here, we have proposed an eco-friendly and cost effective solution for the removal of the heavy metal, Cadmium from industrial effluents. We are pleased to inform you that our biologically developed E.coli system has the ability to regulate growth and protein production, thereby increasing the lifespan of Project Curlim!
Inception of Project CURLIM
Finalization of Strains
Initially, our team worked with two strains of bacteria, Escherichia coli K12 (wild strain) and Escherichia coli DH5ɑ. We performed characterization for Escherichia coli DH5ɑ by checking it’s growth and color change in EMB agar.
E.coli MG1655 was grown in LB broth and E.coli DH5ɑ was grown in Nutrient broth.We observed the growth curve of K12MG1655 and DH5ɑ.After extensive literature review, we decided to move forward with E.coli K12 MG1655.
Improving the Biofilm formation
To identify the biofilm integrity of the native E.coli, we conducted CV assay. We found that the biofilm formed was not strong enough. The biofilm formation was very poor so we decided to enhance the biofilm formation with the help of our construct BBa_K4509469.Since it consists of BBa_J23118 promoter, the burden value is also less and the percentage of growth rate will not be reduced.
Biofilm Formation in Plasmid-induced biofilm
Testing aphA Activity
As mentioned earlier,the main element of our project is acid phosphatase enzyme. The following assays were conducted to check the concentration and activity of the enzyme.
SDS-PAGE
Biuret Test
| Particulars | Blank | Standard Solution | Test | |||||
|---|---|---|---|---|---|---|---|---|
| B | S1 | S2 | S3 | S4 | S5 | T1 | T2 | |
| Volume of working standard (ml) | - | 0.5 | 1 | 1.5 | 2 | 2.5 | - | - |
| Concentration of Working Standard (mg/ml) | - | 2 | 4 | 6 | 8 | 10 | - | - |
| Volume of Test Solution (ml) | - | - | - | - | - | - | 0.5 | 1 |
| Volume of Distilled Water (ml) | 3 | 2.5 | 2 | 1.5 | 1 | 0.5 | 2.5 | 2 |
| Volume of Biuret solution (ml) | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
| Optical density at 520 nm | 0 | 0.03 | 0.07 | 0.1 | 0.16 | 0.18 | 0.01 | 0.05 |
pNPP Assay
1. With enzyme concentration fixed
| Constant Enzyme | ||||||
|---|---|---|---|---|---|---|
| Volume of Citrate Buffer (mL) | Volume of pNPP (mL) | Volume of Enzyme (mL) | Volume of Distilled Water (mL) | Volume of NaOH (mL) | OD at 430nm | |
| 0.5 | 0 | 0.4 | 0.6 | 2.5 | 0 (Blank) | |
| 0.5 | 0 | 0.4 | 0.6 | 2.5 | 0 (Blank) | |
| 0.5 | 0.2 | 0.4 | 0.4 | 2.5 | 0.22 | |
| 0.5 | 0.3 | 0.4 | 0.3 | 2.5 | 0.27 | |
| 0.5 | 0.4 | 0.4 | 0.2 | 2.5 | 0.28 | |
| 0.5 | 0.5 | 0.4 | 0.1 | 2.5 | 0.31 | |
| 0.5 | 0.6 | 0.4 | 0 | 2.5 | 0.34 | |
2. With substrate concentration fixed
| Constant Substrate | ||||||
|---|---|---|---|---|---|---|
| Volume of Citrate Buffer (mL) | Volume of pNPP (mL) | Volume of Enzyme (mL) | Volume of Distilled Water (mL) | Volume of NaOH (mL) | OD at 430nm | |
| 0.5 | 0.4 | 0 | 0.6 | 2.5 | 0 (Blank) | |
| 0.5 | 0.4 | 0.1 | 0.5 | 2.5 | 0.1 | |
| 0.5 | 0.4 | 0.2 | 0.4 | 2.5 | 0.17 | |
| 0.5 | 0.4 | 0.3 | 0.3 | 2.5 | 0.24 | |
| 0.5 | 0.4 | 0.4 | 0.2 | 2.5 | 0.25 | |
| 0.5 | 0.4 | 0.5 | 0.1 | 2.5 | 0.29 | |
| 0.5 | 0.4 | 0.6 | 0 | 2.5 | 0.31 | |
Cadmium Toxicity
As we are using cadmium which is a heavy metal, we performed cadmium toxicity for E.coli. We observed the growth curve of the E.coli under different concentrations of the cadmium.
Control → without cadmium
< MIC(Minimum Inhibitory Concentration) → 1 mg/L
MIC → 2.75 mg/L
< Sub-lethal Concentration → 9.18 mg/L
MTT Assay
The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. The cell viability of the cells were observed after 24 hours using 96 well plates.
<MIC
MIC
<Sub-lethal
Cadmium-E.coli interaction: Morphology
The morphology of the E.coli K12 strain changed drastically in the presence of Cadmium in sub lethal level (36.68mg/l). Figure 8(a) denotes unexposed cells while Figure 8(b) denotes cells exposed to Cadmium.
Figure 9(b)
Atomic Adsorption Spectroscopy
Atomic absorption spectroscopy, is a technique for measuring the concentrations of metallic elements in different materials.Samples were taken at different time intervals to check the concentration of cadmium before the treatment,during the treatment and after the treatment.The result showed significant difference in the concentration of cadmium before and after treatment revealing the efficiency of our project CURLIM!
