Week Seven - 29 July to 4 August


The b. subtilus and c. braaki were taken out of the incubator, parafilmed, and put into the freezer for long term storage.

ECPhy and ECClc were miniprepped.


Prepared for the advisor meeting:
specifically assays and assembly needed lots of work
At the meeting we decided on a phosphate assay that Jenn uses in her lab

Lajja, Sarah, and Tae made ampicillin, tetracycline, and kanamycin stocks


Lajja, Sarah, and Tae made amp/tet/kana plates to use for 3A assembly

Grant and Sarah digested the pSB backbone. Grant and Sarah linearized the pSB1A3 backbone for use in tomorrow’s ligations and transformations.
4 uL of the backbone was digested for 4 hours and then heat killed
7.68 uL of Pbad was digested for 2 hours and then heat killed

Yuan worked on the DNEZ genomic extraction of citrobacter and bacillus subtilus DNA for tomorrow’s PCR of the citrobacter and bacillus phytases. If the DNEZ kit doesn’t work then we will perform a colony PCR.

Tae and Sarah ran a gel on the digested Pbad promoter. It did not appear that the plasmid was cut efficiently as only one bright band was seen on the gel (one small band appeared around 150 nucleotides) Laj
ja and Tae made an overnight from the Pbad stock from last week, attempting to get a colony free of satellites for miniprepping tomorrow


Tae miniprepped the reovernighted Pbad from last night: the concentration of DNA was 59.7 ng/uL

Yuan used the DNEZ kit to extract the genomic DNA from bacillus subtilus. However, Yuan ran out of reagents for the extraction so she did not perform it on citrobacter

Sarah and Grant digested the miniprepped DNA Tae made (with E and S) along with the lysis part (with E and X) along the standard assembly protocol

Tae ran a gel with the digested lysis part and Pb ad(for gel extraction)


Brian transformed pSB1K3 and pSB1T3 (kanamycin and tetracycline backbones for assembly). Brian also transformed the wildtype Pbad promoter after the failure of the strong Pbad promoter during the transformation process


B. subtilus and C. braaki phytases were PCRed out from their respective genomes:
The standard PCR protocols were used, where for colony PCR the addition of a colony was substituted for the addition of DNA
b. subtilus DNA from the DNEZ kit was used in addition to colony PCR
c. braaki DNA could not be obtained from the DNEZ kit due to low amounts of reagants so only colony PCR was used

the amp assembly backbone was PCRed out from the GFP miniprepped part using the primers we ordered
A modified protocol for making linearized backbones was obtained from the registry/last year’s igem team:


In the morning, Tae miniprepped the Kanamycin backbone and pbad
Yuan and Sarah then transformed the Tetracycline backbone