Team:Illinois/Hybrid Promoter

Hybrid Promoter

Goals: The goal of this side-project is the create a hybrid or combinatorial promoter that accepts two inputs. This will be used in the creation of an AND logic gate.

Papers of Interest:

[http://www.nature.com/nature/journal/v420/n6912/full/nature01257.html Engineered gene circuits] Jeff Hasty, David McMillen & J. J. Collins

[http://aem.asm.org/cgi/content/abstract/75/3/637 Construction and Enhancement of a Minimal Genetic AND Logic Gate] Daniel J. Sayut, Yan Niu, and Lianhong Sun

[http://www.sciencemag.org/cgi/content/full/296/5572/1466 Combinatorial Synthesis of Genetic Networks] Cabrevelin C. Guet, Michael B. Elowitz, Weihong Hsing, Stanislas Leibler

[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2132448 Programming gene expression with combinatorial promoters] Robert Sidney Cox, III, Michael G Surette, and Michael B Elowitz

Biobricks of Interest:

BBa_K091101 pTet_Lac hybrid promoter

BBa_R0010 lacl regulated promoter

BBa_R0040 TetR repressible promoter

BBa_K137125 LacI-repressed promoter B4

July 10

We looked more closely at the biobricks: BBa_I14032, BBa_R0040, BBa_K137125, BBa_K091101.

BBa_R0040 and BBa_K091101 were available to us in the registry distribution so we transformed these into cells to test them.

July 13

Today in lab we inoculated a colony of the cells that we transformed the biobricks into yesterday.

Concerns: We want the sRNA gene on a high copy plasmid and the target sequence on low copy plasmid. This could require up to 7 different plasmids to transform into the cell. We need to look into the feasibility of this.

Other things to consider:

- Can we put all of the sRNA expressing genes and their promoters on one plasmid(with all the target sequences and the genes that go with)? would this be too many ligations? How would we tell if they all worked? Run a gel?

- Would we co-transform the plasmids or do them separately?

July 14

In lab we performed a miniprep of the inoculated colony from yesterday to isolate the DNA. It took some time to get access to the centrifuge as it was in use for much of the morning.

At the meeting with the professors they suggested that we take a look at the availabe GFP generator biobricks in the registry. It was also suggested that we would need DH5 alpha Z E. Coli cells if we are to use the lac promoter. It was also mentioned that about two plasmids transformed into a cell was standard and that we could probably get three.

July 15

BBa_I732913 an [aTC] -> RFP measurement biobrick may be useful.

July 17

Attempted a digest of biobricks k113009, R0040, K091101 with EcoRI

July 20

Due to unfortunate circumstances the digestion ran all weekend. Reattempted digestion

July 21

Finished overnight digestion and digested with SpeI

July 22

The gel that was run to inactivate the digestion looked awful.

Reexamined protocol from the fermentas website and decided to attempt a double digest using Spe1 first and then EcoRI

July 24

Used new miniprepped DNA to perform a double digest of K113009, K091101, R0040 with SpeI and EcoRI. We used the following procedure: 1. add components for 20μl reaction: 10μl dH2O, 2μl 10x buffer, ~1μg DNA, 1μl restriction enzyme. 2. mix gently, spin down briefly 3. incubate k@ optimum temp for 1-16 hours The double digest modification required: 1. performing the first digest with 1x Tango buffer and the BcuI enzyme. 2. Incubate for 1 hour 3. Add 10x Tango to a final concentration of 2x Tango buffer (this came out to 2.5μl) 4. Add EcoRI 5. Incubate @ 37C for one hour

July 25

Ran a diagnostic gel to check digests. Ran on a 1% gel at 130v. We don't really use this percent gel and it resulted in a very ugle unreadable gel but it was obvious that the digests did not work.

Reinoculated a colony to attempt again.

July 28

Minipreped the biobrick again, took concentrations. Replated K091101 since there were not many colonies left on the stock plate.

July 30

Transformed biobrick BBa_I20270 in order to isolate the plasmid backbone SB3K3 because earlier efforts the the team to PCR this plasmid have failed.

July 31

Inoculated a colony of I20270 in order to miniprep the plasmid or but we were out of miniprep kits...

August 4

Isolated BBa_I20270 biobrick by miniprep using the Qiagen kit and obtained ~9ng/μl according to the nanodrop.

Started two seperate digestions of the biobrick, one is a double digest and the other is a sequential digest with two different enzymes. We used the fermentas enzymes EcoRI and PstI and used EcoRI first in the single digest. The reaction contained: 10μl DNA, 2μl O buffer, and 2μl of the enzymes (this is more than the 1μl we had been using).

August 5

The digests were stopped by 65C incubation for 20 minutes. The double digest was run on a 2% TAE gel with the Promega 1kb ladder The backbone was extracted from the gel and stored in the -20 freezer. Started the second half of the other SB3K3 digestion at the end of the day

While we believe we have successfully isolated the SB3K3 backbone we are currently unable to ligate it to any promoters with GFP since we do not have and isolated promoters and are currently without the enzymes make them.

August 6

The sequential SB3K3 digestion was taken out and run on a 2% TAE gel but the band was barely visible and is was not possible to extract it.