PROJECT

RESULTS

Day 16 - Tuesday, July 5th 2011

Realized that we could potentially use any of the parts from the registry as plasmid backbones as long as we cut it with the appropriate enzymes and it has a different resistance from the parts that are being ligated.
Transformed the 4 plasmid backbones from the registry, each has a different resistance, placed in incubator overnight.
Gels from last week no longer have visible bands. Tried to restain using ethidium bromide solution, but this did not work.
Miniprepped our parts from overnights we did over the 4th of July weekend to get more DNA
Ran digestion for standard assembly from new miniprep, using all the DNA.
Ran digests on gel.
Unfortunately switched up the gels and ran promoters on the 1.5% gel and the larger parts on the 2.5% gel: once again gel was unsuccessful and we were unable to gel extract
Made overnights and plates from glycerol stocks of all the parts so we can start miniprep again tomorrow.

Day 17 - Wednesday, July 6th 2011

The plates from the overnight stock grew well. The colonies were very dense but we managed to pick single ones to start overnight colonies. We started two overnight cultures for each part so that we can double miniprep and have more DNA to work with.
Only two of our plasmid backbone transformations grew (Amp and Kan). We are unsure why the other transformations may have failed, as our positive controls all grew.
Miniprepped the overnight cultures from last night. The DNA concentrations were better than previous days, but still only in the range of 60-100ng/uL.
We saved a little bit of the miniprepped DNA, but used most of it to digest our parts and run them out on a gel. We used a 1% and 1.5% agarose gel to try and sepearate parts of different sizes.
We retransformed the backbone parts that failed yesterday.
Discussed the idea of using an AND gate to reduce our rate of false positives.

Day 18 - Thursday, July 7th 2011

The overnights looked cloudy and we miniprepped them in the morning.
The tet plasmid backbones grew, but the Chlor ones did not. We also had growth on our retransformations of C0170 and C0171 which got mixed up yesterday.
Searched for parts on the registry that would allow us to implement an AND gate in our project.
Purified all the parts from our gel extraction yesterday and began to ligate several inducible promoter/reporter combinations.
Began another attempt at 3A assembly using the minipreps from this morning and our Kan backbones
Began preparations for another round of competent cell making
Started overnight cultures from yesterday’s transformations
Made glycerol stocks from our successful backbone overnights.
Requested new DNA backbones

Day 19 - Friday, July 8th 2011

Finished up the 3A ligation overnight and transformed bactera with our composite promoter -> reporter parts.
Miniprepped last nights overnight cultures (Two Las parts and the Tet backbone)
Began transforming an RBS part that we will need to add in to most of our parts.
Made SOC and SOB media for our competent cell procedures
Made glycerol stocks of our tet backbones.
Designed primers to extract Las/Rhl promoters from the Pseudomonas Aeruginosa genome.