Team:SDSZ China/Experiment B

iGem SDSZ_China 2018

ABOUT QUORUM SENSING

Quorum sensing is a strategy developed among bacteria that respond to fluctuation of bacteria density in the environment and alters the expression of certain genes. In natural environments, bacteria can control various physiological activities such as bio-illumination, conjugation and so on.

A bacterial QS system (see fig.1)is mediated by what known as the an autoinducer, or a signalling molecule. In low cell density, the autoinducer secreted by individual cells have extremely low density in extracellular environment and are unable to be recognized by receptors and cause significant functional changes in other cells because of the low rate of diffusion. Yet as cell population continues to grow, more autoinducers are secreted and will eventually reach the threshold density. By which point the density is significant enough to activate receptors and began their impact upon cell functions.

As we have already learnt the mechanisms of the system, it’s parts can be used in genetic engineering and help us sorting out problems such as low production efficiency.

WHAT IS CHITOSAN

Because of chitin’s insolubility and bioactivity, it is usually converted by deacetylation to soluble and bioactive CHITOSAN ((C8H13NO5)n (203.19)n). It is white powder and has relatively more industrious usages: Agriculture: used in seed treatment and biopesticide, helping plants to fight off fungal infections. Winemaking: used as fining agent, helping to prevent spoilage. Industry: used in a self-healing polyurethane paint coating. Medicine: used in bandages, helping to reduce bleeding and serving as an antibacterial agent; used to help deliver drugs through the skin.

CURRENT CHITOSAN PRODUCTION

The current technology of chitosan production is the treatment with concentrated alkali. The chitin/chitosan process involves the crushing and drying of crab shell or other suitable species of crustaceans such as shrimp shell waste. The product is processed with acid and alkaline in order to remove protein and calcium. The product is then further dried, grinded, and packaged as a finished or semi-finished product. A plant set-up would involve a number of pieces of equipment for grinding or particuli- zation, drying, acid and alkaline treatment, packaging and effluent treatment. Crushed shrimp waste was kept in a polyethylene bags at ambient temperature (28±2oC) for 24 hours for partial autolysis to facilitate chemical extraction of chitosan and to improve the quality of chitosan. 3steps: Demineralization, Deproteinization and Deacetylation Demineralization: Demineralization of shrimp shell has been carried out with three different concentration of HCI (4%, 3%, 2%) at ambient temperature (28±2oC) with a solid to solvent ratio 1:5 (w/v) for 16 hours (Toan, 2009). The residue was washed and soaked in tap water until neutral pH. Deproteinization: Deproteinization of shrimp shell was done with 4% NaOH at ambient temperature (28±2oC) with a solid to solvent ratio 1:5 (w/v) for 20 hours (Toan, 2009). The residue was washed and soaked in tap water until neutral pH. Then purified chitin was dried until it was become crispy. Chitin flakes was grounded to small particle to facilitate deacetylation. Deacetylation: Removal of acetyl groups from chitin was experimented using four different concentration of NaOH (30%, 40%, 50%, 60%) at 650C temperature with a solid to solvent ratio 1:10 (w/v) for 20 hours. (Toan, 2009).The residue was washed until neutral pH with tap water. The resulting chitosan was then dried at cabinet dryer for 4 hours at 65±50 C and prepared for characterization. However, current chitosan production has exposed many drawbacks: deficiency over time and reactant, huge energy cost during the process, instability of product quality, unsafe condition for employees, and, especially, the destructive pollution of basic waste water. Acid and alkali wasted water can easily polluted waterbody and farmland.