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Revision as of 16:18, 16 September 2010 (view source) Ttodd (Talk | contribs) (New page: {{:Team:BCCS-Bristol/Header}} ==Procedure== How to make them) ← Older edit		Latest revision as of 19:48, 18 October 2010 (view source) Kcoyte (Talk | contribs) (→Procedure)
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	==Procedure==		==Procedure==
-	How to make them	+	To improve signal detection we decided to encapsulate our bacteria inside beads made of gellan gum and set about developing our own procedure. Research yielded several possible options and having experimented with several, we eventually settled modifying a technique developed by Carol Nolan (Nolan, Carol L. (Blue Bell, PA). 1992. Method for microbial immobilization by entrapment in gellan gum. United States Monsanto Company (St. Louis, MO). http://www.freepatentsonline.com/5093253.html)
		+
		+	'''Bead Procedure:'''
		+
		+	It is first necessary to set up a 50mL LB overnight inoculated with a colony of cells you wish to encapsulate, and leave this overnight at 37°C. The next day, follow these steps:
		+
		+	# Add 0.24g of Gellan Gum to 19.6mL of ddH2O. Heat in a waterbath to 90°C, stirring occasionally, then cool to 54°C.
		+	# Meanwhile centrifuge 2 x 25mL overnight at 4500 x g for 8 minutes. Discard the supernatant.
		+	# Wash the pellet in 2mL of ddH2O, resuspend and centrifuge at 4500 x g for 5 minutes. Repeat once.
		+	# Slurry the cells in 0.25mL ddH2O and add to the cooled gum, mix gently by shaking.
		+	# Pipette dropwise into 0.5mL of CaCl2 using the p1000 pipette from approximately 1.5 inches above the surface solution.
		+	# Gently agitate the beads for at least 1 minute then pour off the CaCl2.
		+
		+	This will result in beads approximately 1 - 2mm in diameter.
		+
		+	Note: It is essential to pipette the beads into CaCl2 as soon as possible once the bacteria have been added as the cooler then gellan gum - bacteria mix gets the larger and less uniform the beads formed will be.

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Latest revision as of 19:48, 18 October 2010

Procedure

To improve signal detection we decided to encapsulate our bacteria inside beads made of gellan gum and set about developing our own procedure. Research yielded several possible options and having experimented with several, we eventually settled modifying a technique developed by Carol Nolan (Nolan, Carol L. (Blue Bell, PA). 1992. Method for microbial immobilization by entrapment in gellan gum. United States Monsanto Company (St. Louis, MO). http://www.freepatentsonline.com/5093253.html)

Bead Procedure:

It is first necessary to set up a 50mL LB overnight inoculated with a colony of cells you wish to encapsulate, and leave this overnight at 37°C. The next day, follow these steps:

1. Add 0.24g of Gellan Gum to 19.6mL of ddH2O. Heat in a waterbath to 90°C, stirring occasionally, then cool to 54°C.
2. Meanwhile centrifuge 2 x 25mL overnight at 4500 x g for 8 minutes. Discard the supernatant.
3. Wash the pellet in 2mL of ddH2O, resuspend and centrifuge at 4500 x g for 5 minutes. Repeat once.
4. Slurry the cells in 0.25mL ddH2O and add to the cooled gum, mix gently by shaking.
5. Pipette dropwise into 0.5mL of CaCl2 using the p1000 pipette from approximately 1.5 inches above the surface solution.
6. Gently agitate the beads for at least 1 minute then pour off the CaCl2.

This will result in beads approximately 1 - 2mm in diameter.

Note: It is essential to pipette the beads into CaCl2 as soon as possible once the bacteria have been added as the cooler then gellan gum - bacteria mix gets the larger and less uniform the beads formed will be.