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Team:Valencia/Project2
From 2011.igem.org
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pH-stat: Culture of Synechocystis sp. PCC 6803
Objectives of the culture
General
By introducing a culture of Synechocystis sp cianobaceria. PCC 6803 we intent to ensure that the pH changes resulting from growth and proliferation function as a switch in order to activate the colicins, so that in the artificial system created, as time passes, the colicins produced by Escherichia coli will increase their concentration in their habitat, being inactive until the pH reaches the optimum range of activation, which is when they ´ll get activated and will produce cell lysis and kill the pathogens.
Specific
To develop what we have stated above, we need to know:
• How to establish the culture at the laboratory
• The temporal evolution of the pH in the culture.
Establishing the culture under laboratory conditions.
The material and methods needed are :
• 18 Watts fluorescent tubes of white light, special tubes for aquariums that divide the spectrum mostly between the peaks of the visible red and blue light, which stimulate the photosynthesis.
• Volumetric flasks, 100 ml and 200 ml
• Air Pumps
• Commercial Fertilizer Brand COMPO
• Distilled, tap and analytical water (Type II)
The first experiment consisted of a series of six cultures in which environmental conditions varied so as to know the habitat preferences of the cyanobacterium. We distinguished two different groups:
The first, consisting of C1, C2 and C3, which have tap , distilled and analytical water respectively. It also uses a reflector box, which increases the irradiation of light on the culture, and a magnetic stirrer, which prevents the deposition of cells on the bottom
The second group , which are C4, C5 and C6, without reflector or magnetic stirrer, but we kept the water in the same order as before.
Results and discussion
The other conditions remained constant in both cases, as are detailed in the following table:
The day after having inoculated the culture, the absorbency data were collected and the cells / ml counted with a Neubauer chamber. The absorbancy was measured at 440 nm and 750 nm as the first was the highest value according to the spectrophotometer after a sweep, and the second was taken according to references (Burrows, EH, et. Al., 2009, JF Allen, 2008). In the account we distinguished between simple cells, ie those which are not in the reproductive period, and those which are.
For technical reasons, a power cut for an entire weekend, on our arrival the following Monday, the cultures showed a transparent colour which had nothing to do with their usual blue-green algae colour ( cyanobacteria).So, we got rid of the remains and started a new culture. This time we chose the least contaminated water which still showed a good margin of survival, analytical water, to which we added a higher concentration of fertilizer to stimulate growth. Besides ,we decided to stop using the reflector box as we had evidence that such a bright light caused photo-inhibition. The volumes and other conditions kept constant.
Synechocistys was observed to be viable/ feasible under laboratory conditions. The problem we found is that it is very easy for the culture to get contaminated , appearing unwanted cells pretty soon
La evolución temporal del pH en el cultivo
Se hicieron experimentos para comprobar la variación temporal del pH con los últimos cultivos establecidos, pues son los que mayor concentración hemos obtenido y en los cuales se observa con el microscopio que la mayoría de células están en estado reproductivo. Se impone en el cultivo un nuevo fotoperiodo de 4h/4h, y medimos el pH de éstos 11 veces, 1 cada hora. Obtubimos:
Las variaciones totales del pH son menores de lo esperado en nuestro cultivo, hecho que puede ser debido a que la concentración total de Synechocystis en nuestro cultivo sea inferior a la concentración obtenida con otro medio, como por ejemplo el BG11, medio recomendado para el cultivo de cianobacterias (Portilla, A. et. al., 2009).
Tenemos conocimiento gracias a un grupo colaborador de que las variaciones de pH pueden incrementarse del orden de 1,5 grados en nueve días (Paula Tamagnini, fom IBMC, Porto. Personal comunication. http://www.ibmc.up.pt/index.php?id=447#ibmc). Estas variaciones pueden ser suficientes para activar o inactivar las colicinas.
References
Burrows, E. H., et. al., 2009. Optimization of pH and Nitrogen for Enhanced Hydrogen Production by Synechocystis sp. PCC 6803 via Statistical and Machine Learning Methods. Wiley InterScience. 25: 1009-1018
Allen J.F., et. al., 2008. Evaluation of Acid Stress Tolerance in Synechocystis sp. PCC 6803 Mutants Lacking Signal Transduction-Related Genes sigB, sigD, and rre15Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, 1519–1522.
Portilla, A. et. al., 2009. Evaluación del rendimiento de producción de aceite en cuatro microalgas nativas de las provincias ecuatorianas de Orellana, Esmeraldas, Imbabura y Pichincha. http://www3.espe.edu.ec:8700/bitstream/21000/427/1/T-ESPE-029605.pdf
