Team:BYU Provo/Notebook/Cholera - Enzyme/May-June/Period4/Dailylog

Cholera Enzyme

March-April

May-June

July-August

September

Protocols

Due to the cost of ordering the 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) stain, as well as the safety hazard it presents, we decided to only use CV in our biofilm assay stains. The CV has shown to be adequate for the staining of cholera biofilms and should serve our purposes well.

6/21/13

Our research into possible methods for washing the biofilm in our biofilm assays without disturbing the biofilm all indicated that using a round-bottom well plate is the best way to wash wells with non-adherent cells without significantly disturbing the cells. The round-bottom well plates that could be used in our plate reader are too expensive for us to use, so we will continue looking into alternatives.

We re-attempted the washing of the biofilm in the well plate, but were again unsuccessful in washing the wells without disturbing the biofilm

6/22/13

We prepared a new 96-well plate to test whether adding additional media after 24 hours makes a significant difference in the amount of biofilm growth after 48 hours. As our SLB media gives us significantly better biofilm growth, we will only use this media for all future well plates to give us more consistent data. We prepared the well plate according to the tables below.

6/23/13

We added 50 uL of SLB to rows 2, 4, & 6 of our 96-well plate.

6/24/13

We started an LB overnight with Bacillus subtilis to harvest the bacterial cells. Test tube was left in the 37 degree shaker for 2 days.

We also got access to the RIC Facility (Research Instrumentation Core Facility)that has the plate reader we need. We ran through the programming on the plate reader and set up the experimental procedure for the readings that we will need. Our CV stains will read at 540 nm.

We attempted to run the enzyme treatment on the 96-well plate seeded on 6/21, however in washing the cells the biofilms were significantly disturbed and a significant amount of biofilm was removed in the wash. Using a pipetter on the flat-bottom wells is not working well. We will attempt to use a syringe on the next washes to see if this will help limit the disruption and loss of biofilm during the washes.

6/25/13

We setup restriction digests for AmyA and pJG 648 (pet15b vector). Digests were left overnight in the 37 degree water bath. 30ul of each DNA plasmid was used along with 2ul of each restriction enzyme (BamH1 and Nde1.)

6/26/13

We ran our digest products on gel for AmyA and pet15b. See results below:

[IMAGE]

We cut out gel slices of each products and performed the ligation assay outlined in the Grose Lab Cloning protocol. Ligations were left overnight on desk at room temperature.

We also ran the DNA purification kit on our B. subtilis overnights. We recovered 50 uL purified DNA. We will PCR this DNA tomorrow.

We prepared a new 96-well plate according to the tables below. Plate was placed in 30° incubator for ideal growth conditions.

6/28/13

We treated the 96-well plate according to our treatment protocol listed on 5/13/13 with the exception that only half the number of prescribed washes were performed. This was done in an attempt to limit the disturbance and detachment of the biofilm. We also used 12-gauge needles for the washes to better control the pressure with which the was liquid is expelled into the wells, and the rate at which the liquid is removed. However, during the washes we accidentally punctured the bottom of several of the wells, causing our solution to leak out and compromising the wells. We will start another plate on Monday (7/1).