Part:BBa_K149001:Design

Prp22 promoter

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Biobrick characterization was performed by monitoring promoter activity in different high-stress environments. To perform this experiment, cells were first grown for 24 hours in Yeast Peptone Dextrose (YPD) supplemented with 2% glucose. The following day, cells were transferred to fresh YPD media supplemented with 2% glucose as well as the described concentration of methyl methane sulfonate or sodium chloride. Following 20 hours of growth in each respective environment, cell fluorescence was measured using a BeckmanCoulter FC500 MPL equipped with a 15-mW argon laser. yEGFP excitation was performed at 600v and detected using a 525±15 bandpass filter (FL1).

Figure 1. Flow cytometry graphic comparing yEGFP expression under the PRP22 promoter to auto-fluorescence. The blue population highlights autofluorescence of the cell while prior to SSRE construct integration in BY4741. The population in red illustrates yEGFP expression under the PRP22 promoter when grown in YPD+2% glucose.

Figure 2. Flow cytometry measurements comparing yEGFP expression under the PRP22 when stimulated with methyl methane sulfonate. The blue population highlights yEGFP expression under the PRP22 promoter when grown in presence of 180uM MMS. The population in red illustrates yEGFP expression under the PRP22 promoter when grown in absence of MMS.

Figure 3. Flow cytometry measurements comparing yEGFP expression under the PRP22 when stimulated with methyl methane sulfonate. The blue population highlights yEGFP expression under the PRP22 promoter when grown in presence of 15M NaCl. The population in red illustrates yEGFP expression under the PRP22 promoter when grown in absence of NaCl.

Source

The PRP22 promoter sequence was originally extracted from S. Cervisiae, using the 2 following primers: TAGTAGGATCCATTATTCTGGGCATCCGT;ATACTGAATTCCTCTAATATCTTTGTGTTACCTATGT. A BAMHI restriction site was included within the forward primer, while an EcoRV site was included within the reverse primer. This work was completed by our laboratory technician Simon St-Pierre.

Part Assembly

1. pSB1A2 plasmid containing the BBa_p1010 cell death gene recieved from MIT in ecoli. Miniprep of pSB1A2 plasmid performed.
2. Digestion of the minipreppred pSB1A2 performed using the followong reaction mixture: 25 ul of DNA, 5ul of BSA, 5 ul of Buffer 3, 1 ul EcoRI, 1 ul PstI, 13 ul H2O.
3. pSB1A2 digested for 1 hr at 37C in ecoli incubator.