Team:BYU Provo/Notebook/Cholera - Enzyme/September/Period2/Dailylog

From 2013.igem.org

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<font size="4">'''9/21/13'''</font>
<font size="4">'''9/21/13'''</font>
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We set up our large-scale AmyA Enzyme Assay using the following setup. We will test AmyA at different concentrations and over different time periods to find the best enzymatic response.
+
We set up our large-scale AmyA Enzyme Assay using the following setup. We will test AmyA at different concentrations and over different time periods to find the best enzymatic response. A 4M Urea solution was used for all Urea treatments.
-
 
+
 +
{|class="wikitable" style="text-align:center;"
 +
|+ AmyA Enzymatic Degradation Assay Overnights
 +
|-
 +
!Sample
 +
!Treatment
 +
!Amount of Treatment (uL)
 +
!Time Treatment Added
 +
|-
 +
|1
 +
|rowspan="3" | Untreated
 +
|rowspan="3" | N/A
 +
|rowspan="3" | N/A
 +
|-
 +
|2
 +
|-
 +
|3
 +
|-
 +
|4 || Urea || 50
 +
|rowspan="10" | T = 0 hrs
 +
|-
 +
|5
 +
|rowspan="9" | AmyA
 +
|rowspan="3" | 2.5
 +
|-
 +
|6
 +
|-
 +
|7
 +
|-
 +
|8
 +
|rowspan="3" | 12.5
 +
|-
 +
|9
 +
|-
 +
|10
 +
|-
 +
|11
 +
|rowspan="3" | 25
 +
|-
 +
|12
 +
|-
 +
|13
 +
|-
 +
|14 || Urea || 50
 +
|rowspan="10" | T = 42hrs
 +
|-
 +
|15
 +
|rowspan="9" | AmyA
 +
|rowspan="3" | 2.5
 +
|-
 +
|16
 +
|-
 +
|17
 +
|-
 +
|18
 +
|rowspan="3" | 12.5
 +
|-
 +
|19
 +
|-
 +
|20
 +
|-
 +
|21
 +
|rowspan="3" | 25
 +
|-
 +
|22
 +
|-
 +
|23
 +
|-
 +
|24 || Urea || 50
 +
|rowspan="10" | T = 0 & 42 hrs
 +
|-
 +
|25
 +
|rowspan="9" | AmyA
 +
|rowspan="3" | 2.5
 +
|-
 +
|26
 +
|-
 +
|27
 +
|-
 +
|28
 +
|rowspan="3" | 12.5
 +
|-
 +
|29
 +
|-
 +
|30
 +
|-
 +
|31
 +
|rowspan="3" | 25
 +
|-
 +
|32
 +
|-
 +
|33
 +
|-
 +
|34 || Urea || 50
 +
|rowspan="10" | T = 48 hrs
 +
|-
 +
|35
 +
|rowspan="9" | AmyA
 +
|rowspan="3" | 2.5
 +
|-
 +
|36
 +
|-
 +
|37
 +
|-
 +
|38
 +
|rowspan="3" | 12.5
 +
|-
 +
|39
 +
|-
 +
|40
 +
|-
 +
|41
 +
|rowspan="3" | 25
 +
|-
 +
|42
 +
|-
 +
|43
 +
|}
<br>
<br>

Revision as of 03:46, 23 September 2013


Cholera - Enzymes Notebook: September 15 - September 27 Daily Log



Overview
March-April
May-June
July-August
September

9/16/13

The


9/18/13

We transformed the DspB in the pet15b plasmid into the BL21 strain of competent E. Coli cells. We then set up overnights of Colony 1 of DspB/pIG91 and Colony 1 of AmyA/pIG91.

We also set up overnights of V. cholerae in preparation for setting up and running our Enzyme Assay on Friday in order to test the enzymatic action of AmyA in breaking down V. cholerae biofilms. We will use various amounts of or purified AmyA protein with urea as a positive control and untreated biofilm as a negative control.


9/19/13

We ran plasmid preps for our DspB/pIG91 and AmyA/pIG91 overnights from yesterday using the plasmid prep protocol on the protocol page. The purified plasmids were labeled and placed in the freezer.


9/20/13

We revised our Enzyme Assay protocol today (see Enzyme Assay Revised on protocols page). Rather than growing our biofilms in the 96-well plate, we are going to grow them and run all the treatments in test tubes. In the 96-well plate we were seeing too much variation in growth conditions from the interior of the plate to the exterior as the wells have different air flows and evaporation rates. We will set up our assay to test AmyA enzymatic degradation of V. cholerae biofilms tomorrow.


9/21/13

We set up our large-scale AmyA Enzyme Assay using the following setup. We will test AmyA at different concentrations and over different time periods to find the best enzymatic response. A 4M Urea solution was used for all Urea treatments.

AmyA Enzymatic Degradation Assay Overnights
Sample Treatment Amount of Treatment (uL) Time Treatment Added
1 Untreated N/A N/A
2
3
4 Urea 50 T = 0 hrs
5 AmyA 2.5
6
7
8 12.5
9
10
11 25
12
13
14 Urea 50 T = 42hrs
15 AmyA 2.5
16
17
18 12.5
19
20
21 25
22
23
24 Urea 50 T = 0 & 42 hrs
25 AmyA 2.5
26
27
28 12.5
29
30
31 25
32
33
34 Urea 50 T = 48 hrs
35 AmyA 2.5
36
37
38 12.5
39
40
41 25
42
43


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