Team:BYU Provo/Notebook/Cholera - Enzyme/May-June/Period2/Dailylog

From 2013.igem.org

Cholera - Enzymes Notebook: May 15 - May 31 Daily Log

Cholera Enzyme

March-April

5/15/13

We ran phusion PCR samples on regular agarose gel, but the PCR was unsuccessful. We need to re-setup the PCR products as there was no visible band for either the AmyA or Savinase proteins on our gel.

5/16/13

We reran phusion PCR for AmyA and Savinase. In addition, we also re-boiled the DNA template for Subtilisin savinase. In the PCR master mix we used a GC base thermobuffer in place of the normal 5X Phusion buffer. We hope this will help the PCR reaction for our enzymes.

5/17/13

We ran our phusion PCR products on agarose gel.

Row 1: AmyA control
Row 2: AmyA sample 1
Row 3: Amy A sample 2
Row 4: Savinase control
Row 5: Savinase sample 1
Row 6: Savinase sample 2

The AmyA products worked. There was no visible band for the Savinase products. It seems like we are not getting adequate DNA from our lysed Bacillus Subtilis cells. We need to determine a new boiling method to better isolate the Savinase DNA in preparation for the PCR.

5/20/13

We set up our first 96-well plate for the biofilm assay. We want to determine the best incubation time for the plate to have sufficient biofilm growth for us to run the assay once the enzymes are ready. For our initial determination, we set up the plate as shown in the tables below. The plate was placed in the 30 °C incubator as our biofilm shows the strongest growth at this temperature.

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	2.4	24	1
3-4	LB	2.4	48	2
5-6	LB	2.4	96	4
7-8	SLB	2.4	24	1
9-10	SLB	2.4	48	2
11-12	SLB	2.4	96	4

5/21/13

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.

5/22/13

Today we again added 50 ul of each media to ensure biofilm growth restoration. The media was carefully replaced by dispensing the media along the side of each well.

Future plans:

On 5/24 make new SSW LB and see new cholera overnights
Design outline for childrens book for IGEM outreach project
On 5/27 reset up biofilm assays and change the media 5/28
5/29 complete run through of designed biofilm assays
Set up restriction digests of AmyA for cloning procedure

5/24/13

We prepared 500 mL of new SLB media following the recipe listed on 4/3/13. The exact measurements for salt added to the synthetic sea water were:

12.04 g NaCl

5.54 g MgCl2

0.99 g CaCl2

0.41 g KCl

5/29/13

We set up AmyA cloning with plasmid pIG 86 and pet 15b vector (pJG 648) for cloning. We set up restriction digests with the following protocol, using BamH1 and Nde1 as our restriction enzymes:

14ul ddH2O

5ul buffer 4

0.5ul BSA

30ul DNA plasmid

1.5ul of each restriction enzyme

We left the restriction digests overnight in the 37° water bath.

Today we received the Dispersin B plasmid (iGem part BBa_K802001) that we had requested from the Lyon-INSA iGem Team, who were working with this part last year. The sample was inadequately packed in an eppendorf inside of a plastic test tube. The test tube was shattered and the eppendorf was cracked along the side. We performed an E. coli transformation for Dispersin B and added 10ul to the eppendorf to attempt to rescue the DNA sample. We then transferred this into DH5-alpha cells and followed E. coli TFN protocol. TFN was plated on LB-AMP and left in 37° incubator overnight.

5/30/13

TFN for DspB was unsuccessful; we were unable to recover any of the sample. We requested another sample of iGem part BBa_K802001 from the Lyons-INSA iGem team, hopefully this sample will arrive intact.

5/31/13

Ran pet15b vector and AmyA restriction digests on low melt gel. There were no digest products for AmyA. We need to reset up the digest and recheck the restriction enzymes.