Difference between revisions of "Part:BBa K2966006"

Revision as of 03:46, 22 October 2019

PpsbA-NodC_Pf-T4 term

This part is the constitutive promoter, Pc along with the codon-optimized NodC for P. fluorescens and finally the T4 transcriptional terminator. The expected band size is 1.2 kb.

Characterization

Calcofluor white is a stain that fluoresces blue when in the presence of chitin and UV light. As seen below, colonies with that contain a gene expressing chitin (left) fluoresce blue much brighter than the wildtype strain. The calcofluor white assay was used to determine if chitin was being produced by our cells. Both a blue and a white colony were selected from the pGEM plate, and it was assumed that the blue colony would not have the gene inserted and the white colony would. Each colony was suspended in 200 microliters of PBS, and then 20 microliters of calcofluor white was added to the tube. The cells then sat for one minute, and then 40 microliters were pipetted onto a microscope slide. Once the glass coverslip was put on, the slide was analyzed under a fluorescent microscope. The cells were observed on a normal light setting and with UV light, and then an overlay image was created to compare those two views.

The figure below shows Pc with NodC_Pf, a negative control, and PpsbA with NodC_Pf. The negative control was used to show that the wildtype E. coli does not normally fluoresce. Pc with NodC_Pf had almost all of its cells fluoresce blue, and PpsbA with NodC_Pf had most of its cells fluoresce as well.

50px Sequence and Features