Team:Illinois/Hybrid Promoter

From 2009.igem.org

(Difference between revisions)
Line 86: Line 86:
[[Image:UI09_DR_promoter_digestion_7-22.jpg|200px|thumb|center]]
[[Image:UI09_DR_promoter_digestion_7-22.jpg|200px|thumb|center]]
 +
 +
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.

Revision as of 21:27, 24 July 2009

Click to go to the Illinois home page



Contents

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:

Engineered gene circuits Jeff Hasty, David McMillen & J. J. Collins

Construction and Enhancement of a Minimal Genetic AND Logic Gate Daniel J. Sayut, Yan Niu, and Lianhong Sun

Combinatorial Synthesis of Genetic Networks Cabrevelin C. Guet, Michael B. Elowitz, Weihong Hsing, Stanislas Leibler

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

Today we came up with a schematic of a possible decoder:
IllinoisDecoderjuly10.png


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.

UI09 DR promoter digestion 7-22.jpg

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.


Questions about our Wiki page? Please email us at illinoisiGEM@gmail.com.