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

From 2013.igem.org

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(Created page with "{{TeamBYUProvo}} <br> {| width="100%" | colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Cholera - Enzymes Notebook: June 15 - June 30 Daily Log'''</font> ...")
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<font face="Calibri" size="3">
<font face="Calibri" size="3">
-
<font size="4"> '''6/15/13''' </font>
+
<font size="4"> '''6/19/13''' </font>
-
Insert Text
+
We examined our cholera overnights and found that the overnights seeded on 6/7 still have strong biofilms. The overnights seeded on 6/17 are already showing biofilm growth, however another day would be ideal for the highest biofilm growth.
 +
 
 +
We prepared the 0.3% CV solution for our biofilm assay protocol and prepared a new 96-well plate according to the following tables.
 +
 
 +
{|class="wikitable" style="text-align:center;
 +
|+ 96-Well Plate Setup
 +
|-
 +
!Treatment:
 +
!
 +
!Blank Control
 +
!Biofilm
 +
!Urea
 +
!Biofilm w/ CV
 +
!Urea w/ CV
 +
!
 +
!
 +
|-
 +
!Column:
 +
| H || G || F || E || D || C || B || A
 +
|}
 +
 
 +
{|class="wikitable" style="text-align:center;"
 +
|+ Master Solutions
 +
|-
 +
!Row
 +
!Growth Media
 +
!Amount of Media (mL)
 +
!Amount of V. cholerae (uL)
 +
!Percent V. cholerae
 +
|-
 +
| 1 || LB || 1.2 || 12 || 1
 +
|-
 +
| 2 || LB || 1.2 || 24 || 2
 +
|-
 +
| 3 || LB || 1.2 || 48 || 4
 +
|-
 +
| 4 || SLB || 1.2 || 12 || 1
 +
|-
 +
| 5 || SLB || 1.2 || 24 || 2
 +
|-
 +
| 6 || SLB || 1.2 || 48 || 4
 +
|}
 +
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.
<br>
<br>
-
<font size="4"> '''6/16/13''' </font>
+
<font size="4">'''6/21/13'''</font>
-
Insert Text
+
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
 +
 
 +
<br>
 +
 
 +
<font size="4">'''6/22/13'''</font>
 +
 
 +
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.
 +
 
 +
{|class="wikitable" style="text-align:center;"
 +
|+ 96-Well Plate Setup
 +
|-
 +
!Treatment:
 +
!
 +
!Blank Control
 +
!Biofilm
 +
!Urea
 +
!Biofilm w/ CV
 +
!Urea w/ CV
 +
!
 +
!
 +
|-
 +
!Column:
 +
| H || G || F || E || D || C || B || A
 +
|}
 +
 
 +
{|class="wikitable" style="text-align:center;"
 +
|+ Master Solutions
 +
|-
 +
!Row
 +
!Growth Media
 +
!Amount of Media (mL)
 +
!Amount of V. cholerae (uL)
 +
!Percent V. cholerae
 +
|-
 +
| 1 || SLB || 1.2 || 12 || 1
 +
|-
 +
| 2 || SLB || 1.2 || 12 || 1
 +
|-
 +
| 3 || SLB || 1.2 || 24 || 2
 +
|-
 +
| 4 || SLB || 1.2 || 24 || 2
 +
|-
 +
| 5 || SLB || 1.2 || 48 || 4
 +
|-
 +
| 6 || SLB || 1.2 || 48 || 4
 +
|}
 +
 
 +
<br>
 +
 
 +
<font size="4">'''6/23/13'''</font>
 +
 
 +
We added 50 uL of SLB to rows 2, 4, & 6 of our 96-well plate.
 +
 
 +
<br>
 +
 
 +
<font size="4">'''6/24/13'''</font>
 +
 
 +
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.
 +
 
 +
<br>
 +
 
 +
<font size="4">'''6/25/13'''</font>
 +
 
 +
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.)
 +
 
 +
<br>
 +
 
 +
<font size="4">'''6/26/13'''</font>
 +
 
 +
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.
 +
 
 +
{|class="wikitable" style="text-align:center;"
 +
|+ 96-Well Plate Setup
 +
|-
 +
!Treatment:
 +
!
 +
!Blank Control
 +
!Biofilm
 +
!Urea
 +
!Biofilm w/ CV
 +
!Urea w/ CV
 +
!
 +
!
 +
|-
 +
!Column:
 +
| H || G || F || E || D || C || B || A
 +
|}
 +
 
 +
{|class="wikitable" style="text-align:center;"
 +
|+Master Solutions
 +
|-
 +
!Row
 +
!Growth Media
 +
!Amount of Media (mL)
 +
!Amount of V. cholerae (uL)
 +
!Percent V. cholerae
 +
|-
 +
| 1 || SLB || 1.2 || 12 || 1
 +
|-
 +
| 2 || SLB || 1.2 || 12 || 1
 +
|-
 +
| 3 || SLB || 1.2 || 24 || 2
 +
|-
 +
| 4 || SLB || 1.2 || 24 || 2
 +
|-
 +
| 5 || SLB || 1.2 || 48 || 4
 +
|-
 +
| 6 || SLB || 1.2 || 48 || 4
 +
|}
 +
 
 +
<br>
 +
 
 +
<font size="4">'''6/28/13'''</font>
 +
 
 +
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).
 +
 
 +
<br>
</font>
</font>

Revision as of 05:35, 6 September 2013


Cholera - Enzymes Notebook: June 15 - June 30 Daily Log



Overview
March-April
May-June
July-August
September-October

6/19/13

We examined our cholera overnights and found that the overnights seeded on 6/7 still have strong biofilms. The overnights seeded on 6/17 are already showing biofilm growth, however another day would be ideal for the highest biofilm growth.

We prepared the 0.3% CV solution for our biofilm assay protocol and prepared a new 96-well plate according to the following tables.

96-Well Plate Setup
Treatment: Blank Control Biofilm Urea Biofilm w/ CV Urea w/ CV
Column: H G F E D C B A
Master Solutions
Row Growth Media Amount of Media (mL) Amount of V. cholerae (uL) Percent V. cholerae
1 LB 1.2 12 1
2 LB 1.2 24 2
3 LB 1.2 48 4
4 SLB 1.2 12 1
5 SLB 1.2 24 2
6 SLB 1.2 48 4

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.

96-Well Plate Setup
Treatment: Blank Control Biofilm Urea Biofilm w/ CV Urea w/ CV
Column: H G F E D C B A
Master Solutions
Row Growth Media Amount of Media (mL) Amount of V. cholerae (uL) Percent V. cholerae
1 SLB 1.2 12 1
2 SLB 1.2 12 1
3 SLB 1.2 24 2
4 SLB 1.2 24 2
5 SLB 1.2 48 4
6 SLB 1.2 48 4


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.

96-Well Plate Setup
Treatment: Blank Control Biofilm Urea Biofilm w/ CV Urea w/ CV
Column: H G F E D C B A
Master Solutions
Row Growth Media Amount of Media (mL) Amount of V. cholerae (uL) Percent V. cholerae
1 SLB 1.2 12 1
2 SLB 1.2 12 1
3 SLB 1.2 24 2
4 SLB 1.2 24 2
5 SLB 1.2 48 4
6 SLB 1.2 48 4


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).


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