Will Notebook1

Back to Main Page

Wideloache 14:39, 23 September 2008 (EDT)

I ran the digestions on a .8% gel, and obtained the following result:

1=S03954(E/S), 2=B0015(E/X), 3=J23100(S/P), 4=S03983(X/P)

make freezer stocks of:
S03954
J23100
B0015
Ec100D::pir116 cells from Chris

Wideloache 13:58, 22 September 2008 (EDT)

Last night I grew up S03983 (LasR-TT) in LB. Today I did a Zyppy MP and obtained a concentration of 139.9 ng/uL. I then performed the following digestions:

13 uL H20
3 uL DNA
2 uL Buffer
1 uL RE 1
1 uL RE 2

S03954 -> Front Insert (E/S) + B0015 -> Front Vector (E/X)

J23100 -> Back Vector (S/P) + S03983 -> Back Insert (X/P)

This is the first of two rounds of ligation need to make the construct: Prom-LuxR-TT-Prom-LasR-TT. This will be inserted into the genome using the plasmids shipped from the Anderson Lab last week.

Wideloache 14:44, 19 September 2008 (EDT)

I obtained minipreps of B0015, J23100, and S03954 (Promoter-LuxR) from the iGEM racks. I couldn't find a miniprep of S03983 (LasR-TT), but I got a sample from the -80. Note: S03954 was not in the -80 as far as I could tell. I will freeze this down along with the other MP's after I transform them into cells.

I also received the shipment from Chris and my oligos were shipped today. I will begin with pLas assembly and LuxR/LasR genome insertions this weekend.

Wideloache 14:03, 18 September 2008 (EDT)

Over the past few days, I have been getting up to speed on the project as a whole. A lot of time was spent figuring out the primer sequences for the pLux' and pLuxLas promoters. I found that the primers that had been ordered over the summer used the -10 region from the registry, which differed from the standard consensus sequence in E. coli. I also found that a tube switch had occurred over the summer since the sequences for pLux' matched the expected sequence for pLuxLas and vice versa. Still, all 6 clones had at least one mutation in them. I decided to order new primers with the consensus -10 region. This only required that I reorder the reverse primers. I will reuse the forward primers that were designed over the summer.

I also contact Chris Anderson about performing a genomic insertion of LuxR and LasR. He is in the process of shipping the necessary plasmids to me. I will be doing a phi80 genome integration (protocol).

Wideloache 16:20, 10 September 2008 (EDT)

On Monday, I got K091136, K091146, and K091117 out of the -80. I need to make pLux' and pLux+Las- and do a genomic insertion of LuxR/LasR before I can test the XOR promoters.

Begin Primer Synthesis of pLux' and pLux+Las- (could consider site directed mutagensis with Pallavi's primers)
Email Chris about genomic insert vector
Talk to Erin Feeney about the status of the LuxR and LasR cassettes