Team:BYU Provo/Notebook/Cholera - Enzyme/May-June/Period3/Dailylog

From 2013.igem.org

Cholera - Enzymes Notebook: June 1 - June 14 Daily Log

Cholera Enzyme

March-April

6/1/13

We set up a 96-well plate for a trial biofilm assay according to the tables shown below. The well plate was placed in the 30° incubator overnight.

96-Well Plate Setup
Treatment:	Blank Control	Biofilm	Urea	Urea w/ CTC	Urea w/ CV	Biofilm w/ CTC	Biofilm 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
1-2	LB	2.8	28	1
3-4	LB	2.8	56	2
5-6	LB	2.8	112	4
7-8	SLB	2.8	28	1
9-10	SLB	2.8	56	2
11-12	SLB	2.8	112	4

6/2/13

We added 50 ul of media to their respective wells to prevent evaporation of the samples. The media was carefully added by dispensing the media along the side of each well. The plate was placed back in the 30° incubator.

6/3/13

We started new overnights of v. cholerae (two from previous overnights, two from plate) and placed them in the 30° incubator.

We also researched methods to isolate the genomic DNA of bacillus subtilis in order to extract the gene for subtilisin savinase. To do this, we obtained a PureLink Genomic DNA mini kit from the Johnson lab at BYU and will follow the outlined procedure to isolate the bacterial DNA of b. subtilis. We started a 1 mL overnight of b. subtilis in regular LB media broth and left in the 37° shaker overnight.

We also attempted a test run with our biofilm assay to wash the biofilm per the protocol without significantly disturbing the biofilm The attempt was not successful, any attempt to wash the biofilm caused complete disruption of the biofilm and detachment from the well walls. We will research this problem further to see if we can find a solution.

6/5/13

Our SLB was contaminated so we made 500 mL of new SLB. We also obtained fresh Lysozyme to be able to prepare the Lysozyme buffer needed for the PureLink Genomic DNA mini kit to purify/extract DNA from bacillus subtilis.

We also heard back from the Lyons-INSA iGem team. They are currently prepping for finals and will be unable to send us a sample of iGem part BBa_K802001 for about a month. Once they are finished with finals we can contact them again to have them send the part. We are looking into other teams that we might be able to get this part from.

6/7/13

We prepared our lysozyme buffer using the following protocol:

The final buffer should be:

25 mM Tris-HCl - pH 8.0

2.5 mM EDTA

1% Triton X-100

To create 25 mL total buffer volume, we used:

0.0985 g Tris-HCl

0.125 mL EDTA (0.5 M)

0.25 mL Triton X-100

To 200 uL of the buffer, we added fresh lysozyme to obtain a final concentration of 20 mg/mL lysozyme.

We also streaked a new cholera plate and prepped new cholera overnights.

6/10/13

Today we ran the PureLink Genomic DNA mini kit on our bacillus subtilis.

6/12/13

We ran phusion PCR for AmyA and Savinase. PCR master mix included the GC thermobuffer.

Primers used:

AmyA - BI259 (f), BI260 (r)

Savinase - BI255 (f), BI256 ®

Labelled PCR Tubes:

A1 - AmyA w/ template DNA

A2 - AmyA control

S1 - Savinase w/ template DNA

S2 - Savinase control

6/14/13

Ran PCR products on regular agarose gel. See results below: [IMAGE]

The AmyA product is visible. We will run the clean up kit on this on Monday. There was no visible band for the Savinase. We need to rerun the Genomic DNA kit with Savinase. We could try adding 1 mL DMSO to help with the difficulty of the phusion PCR for Savinase.

We also obtained 5 g crystal violet from Dr. Michaelis’ lab in the Chemistry department. We will prepare our 0.3% CV solution for our biofilm assay protocol on Wed (6/19).