The goal of our team is to design a cadmium biosensor in the form of a dipstick that will glow red (express RFP) in the presence of cadmium. This biosensor will help us collect data from soil samples and determine if a bioremediation process should be started. Then, with genetically modified plants, we plan to hyperaccumulate the cadmium in the soil. For the design of our biosensor we have been using PCadA (BBa_K1724000) as our cadmium sensitive promoter. However, one of the parts that could help in the future of our project is BBa_K1342005, the ZinT gene with a cadmium sensitive promoter.
The following has been added to the Parts Registry page of BBa_K1342005:
Bibliography
Hobman, J. (2015, July 7). Zinc dependence of Zint (YODA) mutants and binding of zinc, cadmium and Mercury by Zint. Biochemical and Biophysical Research Communications. Retrieved October 10, 2022, from https://www.academia.edu/13736354/Zinc_dependence_of_zinT_yodA_mutants_and_binding_of_zinc_cadmium_and_mercury_by_ZinT
Ferianc, P., Farewell, A., & Nyström, T. (1998, April 1). The Cadmium-Stress Stimulon of Escherichia Coli K-12. Microbiology. Retrieved October 10, 2022, from https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-144-4-1045?crawler=true
EcoCyc. (2022, June 21). Metal-binding Protein ZinT. Escherichia coli K-12 substr. MG1655 Zint. Retrieved October 11, 2022, from https://ecocyc.org/gene?orgid=ECOLI&id=G7061#
To achieve our goal of using DH5 alpha Escherichia coli as a cadmium biosensor, and later, hopefully as a cadmium bioaccumulator, we first needed to ensure the survival of E. coli at certain cadmium concentrations that we were going to put the E. coli in. Hence, we decided to investigate E. coli resistance to CdSO4 at different concentrations. The Kirby Bauer method (otherwise called the zone of inhibition method) was used for this experiment, where we grew 5 plates with the 5 dics soaked with CdSO4 solutions at concentrations 0mM, 0.3mM, 0.6mM, 0.9mM, and 1.2mM. The plates were kept in the incubator at 28 Degrees Celsius for 48 hours. We hypothesized that we would get small, yet increasing zones of inhibition at concentrations from 0mM to 0.9mM, and a relatively larger zone of inhibition for 1.2mM.
Why did we choose these specific concentrations?
In the parts registry of BBa_K1724000, we noticed that the team used 0.00003mM, 0.0001mM, 0.001mM, 0.01mM, 0.1mM as their cadmium ions concentration to test their cadmium biosensor with pcadA, a promoter that our team is also aiming to use in the future. Hence, we were curious to see if the E. coli is able to survive at higher concentrations than the ones tested in the registry. Additionally, “E. coli is intrinsically tolerant to high levels of cadmium up to 0.9–1.0 mM” (Qin et al.). Subsequently, we decided to test cadmium resistance of E. coli at final concentrations of 0mM, 0.3mM, 0.6mM, and 1.2mM.
(figure 1. One of the streaked plates for the experiment)
From our results, we found that DH5 alpha E. coli was resistant to cadmium concentrations from 0.3 to 0.9mM, as no zones of inhibition were identified. We also found that the bacteria was tolerant to 1.2mM of CdSO4 as well, and small colonies were found. This suggests that E. coli could tolerate cadmium concentrations up to 1.2mM, which is 0.2mM more than what (Qin et al.).suggested. Hence, we were able to conclude that we are able to experiment with DH5 alpha E. coli at cadmium concentrations up to 1.2mM for cadmium detection and accumulation without having to consider the survival of E. Coli influencing our results.
Furthermore, the average concentration of cadmium in Peruvian soil in cacao farms ranges between “1.02 and 3.54 mg kg−1” (Oliva et al.). When we test our biosensors, we are planning to create solutions out of the soils collected from contaminated cacao farms. Our estimated highest cadmium concentration would be created from 50g of extracted soil and pouring in 100ml of dH2O (subject to change). This would then create a solution ranging from approximately 4.54*10-6 mM to 1.57*10-2 mM of cadmium concentration. Hence, E. coli surviving in our experimental concentrations from 0 mM to 1.2 mM would imply that our bioengineered biosensors would also survive in cadmium contaminated soil in Peru, further meaning that implementing our proposed idea from this project would be reasonable in peruvian cacao plantation industries.
This may be beneficial for our future project and other iGem teams who aim to experiment E. coli biosensors and/or bioremediation of cadmium since the survival of the bioengineered E. coli would be crucial to carry out the programmed function properly and achieve the goal that we/ any other team working with E. coli & cadmium are striving for.
The following has been added to the Parts Registry page of BBa_K1724000:
Bibliography
Oliva, Manuel, et al. “Cadmium Uptake in Native Cacao Trees in Agricultural Lands of Bagua, Peru.” Agronomy, vol. 10, no. 10, Oct. 2020, p. 1551. Crossref, doi:10.3390/agronomy10101551.
Qin, Weitong, et al. Identification of Cadmium Resistance and Adsorption Gene from Escherichia Coli BL21 (DE3) - RSC Advances (RSC Publishing) DOI:10.1039/C7RA10656D. Royal Society of Chemistry, 6 Nov. 2017, pubs.rsc.org/en/content/articlehtml/2017/ra/c7ra10656d.