Difference between revisions of "Team:IISER-Mohali-INDIA/Co-culture"

Co-culturing is a technique of culturing or growing two or more than two different populations of cells in the same culture plate. It is a useful way to understand the interaction between two different types of cells, making it advantageous for studies of cell-cell communication.

Here in we have the plan to co-culture our engineered strain of E. coli (which can independently detect and capture each of the noxious gases and harmful chemicals) with naturally occurring microbes which can consume these harmful gases and chemicals through their evolved metabolic pathways. This ensures the existence of a sink for these pollutants, and thereby the longevity of the synthetic microbes.

Simply introducing the metabolic pathways of qualified co-culture cells (which can consume these harmful gases and chemicals) in a synthetic microbe induces immense burden and reduces its longevity. This approach has been taken up by many teams, only to result in the non-viability of these microbes. Therefore, we have taken a novel co-culturing method which is partially synthetic and partially natural to tackle pollution in a more efficient and effective manner.

The study of bacterial growth has been one of the most standard approaches in the field of microbiology and Escherichia coli as a workhorse has been considerably exploited for its use in many molecular biology techniques. The need of a nutrient rich solid or liquid medium is of utmost importance for the sustenance of bacterial growth. However, people over the past few years have tried using paper based growth supports for bacteria. Paper is a ubiquitous, inexpensive, recyclable, portable, flexible and disposable material. But paper can’t be directly used for culturing microbes due to certain constraints such as the requirement of a nutrient rich medium for growth, sample dispersion and absorption by the paper, etc. Hence, paper needs to be synthetically modified for sustaining the growth of microbes. The construction of in-vitro biological systems is a remarkable area of study in synthetic biology and has been widely exploited and implemented for this approach. These systems make use of strong promoters and translation initiation sequences for the expression of most of the individual genes encoded on the genome of interest. In this, the microbial culture after having been grown to a suitable density, is subjected to lysis and the lysate is added to the synthetically modified paper and subjected to freeze drying. At the time of use, the components on the paper can be revived by adding distilled water or a suitable buffer and heating the paper at an optimum growth temperature followed by adding the components (compatibility of the components used for the reaction is a very important factor) required for transcription-translation and the required reaction can hence be carried on the paper. Fine tuning of the circuit and improvisation in the design of the paper being used for the reaction needs to be taken care of to improve the efficiency of the reaction.

It has been already documented that bacteria culture can be grown on paper even in a cordinated manner with the help of inkjet paper [1]. To do this, paper is completely immersed in toluene solution of polystyrene and then completely drying for making the hydrophobic surface [1]. Then, small patches are made by inkjet paper with toluene to form hydrophilic patches [1]. Agar is hydrolysed by sulphuric acid for appropriate viscosity and dispensed with inkjet printer [1]. Sodium hydroxide can be used to neutralise the agar and sodium sulphate can be removed by electrodialysis [1]. The agar is printed on the hydrophilic patches and can be used to grow bacteria [1]. The paper is folded in a specific manner to be accommodated inside the device as shown in the image.

References:

1. Srimongkon, T., Ishida, T., Igarashi, K., & Enomae, T. (2014). Development of a bacterial culture system using a paper platform to accommodate media and an ink-jet printing to dispense bacteria. Am. J. Biochem. Biotechnol, 10, 81-87.

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5. Wang, X., Wang, Q., Li, S., & Li, W. (2015). Degradation pathway and kinetic analysis for p-xylene removal by a novel Pandoraea sp. strain WL1 and its application in a biotrickling filter. Journal of hazardous materials, 288, 17-24.

6. Diekert, G. B., &Thauer, R. K. (1978).Carbon monoxide oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum. Journal of bacteriology, 136(2), 597-606.

7. Chappelle, E. W. (1962). Carbon monoxide oxidation by algae. Biochimicaetbiophysicaacta, 62(1), 45-62. Slomczynski, D. J. AND W J. DavisHoover*.CHARACTERIZATION OF PB2+ UPTAKE AND SEQUESTRATION IN PSEUDOMONAS AERUGINOSA, CHL004, LEAD.Presented at Third International Conference, Monterey, CA, 5/20-23/2002.

8. Wolfe-Simon, F., Blum, J. S., Kulp, T. R., Gordon, G. W., Hoeft, S. E., Pett-Ridge, J., ...& Anbar, A. D. (2011). A bacterium that can grow by using arsenic instead of phosphorus. science, 332(6034), 1163-1166.