Difference between revisions of "Part:BBa K2966007"
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<h2>Characterization</h2> | <h2>Characterization</h2> | ||
| − | 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 | + | 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 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. | + | The figure below shows Pc with NodC_Pf, a negative control, and PpsbA with NodC_Pf in that exact order. 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. |
https://2019.igem.org/wiki/images/b/b8/T--Purdue--S3controlS4.jpg | https://2019.igem.org/wiki/images/b/b8/T--Purdue--S3controlS4.jpg | ||
Latest revision as of 03:49, 22 October 2019
Pc-NodC_Pf
Pc, a constitutive promoter with the codon-optimized NodC for P.fluorescens.
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 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 in that exact order. 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 52
- 1000COMPATIBLE WITH RFC[1000]