Team:Purdue/Results
PGEM:
Our plasmids were transformed into JM109 cells and ligated into the pGEM vector to determine if the gene of interest was inserted correctly. The bacteria were then plated onto an LB plate with X-gal, IPTG, and Ampicillin so that the colonies would grow to either be blue or white- the white colonies were selected from these plates for pure cultures to be made from. The presence of the gene product would then be later confirmed with an enzyme digest.
Our plasmids were transformed into JM109 cells and ligated into the pGEM vector to determine if the gene of interest was inserted correctly. The bacteria were then plated onto an LB plate with X-gal, IPTG, and Ampicillin so that the colonies would grow to either be blue or white- the white colonies were selected from these plates for pure cultures to be made from. The presence of the gene product would then be later confirmed with an enzyme digest.
Agarose Gel images:
Plasmids isolated from white colonies on the pGEM were digested with EcoR1 to confirm the presence of desired sequences in the pGEM backbone. Gel electrophoresis was performed to visualize the bands. If the band appeared in the correct location, then the miniprepped DNA was sent off for sequencing as the last confirmatory step.
Plasmids isolated from white colonies on the pGEM were digested with EcoR1 to confirm the presence of desired sequences in the pGEM backbone. Gel electrophoresis was performed to visualize the bands. If the band appeared in the correct location, then the miniprepped DNA was sent off for sequencing as the last confirmatory step.
SDS Page:
An SDS Page gel was used to visualize and identify the protein molecules produced by Gold 1. The gel shown below confirmed the expression of the 20 kDa NodC protein with the SDS page
An SDS Page gel was used to visualize and identify the protein molecules produced by Gold 1. The gel shown below confirmed the expression of the 20 kDa NodC protein with the SDS page
Calcofluor White Stain:
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 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.
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 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 above 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
The figure shown above shows NodC with an Anderson on the top and a negative control from the same plate on the bottom. The chitin product was shown to be produced since the cells fluoresce after being mixed with the calcofluor white assay. The control was used as a comparison to show how wildtype E. coli does not have most of its cells fluoresce.
