Part:BBa_K1225000
PART DESCRIPTION: Constitutive promoter
This is a medium-strength Escherichia coli Ptrc* promoter. The asterisk denotes the lack of an operator downstream of the transcription start site, -35 to +1 of the promoter. The promoter's sequence comes from "Precise and reliable gene expression via standard transcription and translation initiation elements" by Vivek K. Mutalik and colleagues. Two inward pointing BpiI (BbsI) sites were added between the original promoter sequence and the BioBrick prefix and suffix to allow seamless assembly into the 2013 Purdue iGEM team's Bicistronic Design (BCD) Expression Operating Units, modified versions of BCDs 7, 18, 22 and 23 created by Mutalik et al. in the aforementioned work.
Contact Information
Author(s): James Nolan and Chris Thompson
Team: Purdue University 2013
Data Collection: Amanda Shanley, Chris Thompson, and James Nolan
Affiliation: Purdue University (Bindley Bioscience Center)
Contact: nolan6@purdue.edu
Related Parts: None
Date: 09/27/13
Standard Design Information
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Chassis: E.coli
Strain: BL21
Device Name: BBa_K1225000
Device Type: Promoter
Safety Level: Risk Group 1
Assembly: Golden Gate Assembly
Protocol: Freiburg Golden Gate Protocol
Scars: None
Insertion: Plasmid
Vector: pSB1C3
Growth Rate Assay
BASIC INFORMATION
Purpose: To assess what effect, if any, our genetic parts have on the growth rate of E.coli.
Chassis: E.coli
Strain: BL21
Protocols: Purdue iGEM Growth Curve Protocol
Date: 09/25/13
GROWTH CONDITIONSMedia Type: Luria Broth (LB)
Vessel: 10mL Culture Tube
Volume: 5mL
Incubation: 37 C, 250 rpm
MEASUREMENT INFORMATION
Data Type: Growth Curve (OD vs Time)
Location: Bindley Bioscience Center
Machine Name: N/A
Time Interval: 30min
Total Time: 270min
Proof of Functionality
BASIC INFORMATION
Purpose: To qualitatively and quantitatively prove that our promoter initiates transcription by placing mRFP after it.
Chassis: E.coli
Strain: BL21
Protocols: None
Date: 09/26/13
GROWTH CONDITIONSMedia Type: Luria Broth (LB)
Vessel: 10mL Culture Tube
Volume: 5mL
Incubation:37 C, 250 rpm
MEASUREMENT INFORMATION
Data Type: Fluorescence Intensity vs Wavelength
Location: Bindley Bioscience Center
Machine Name: Cary Eclipse
Excitation: 584nm
Emission: 600nm-620nm
Notes: In the first graph, a plot of intensity vs wavelength is shown. The prtc* promoter was inserted into three separate bicistronic design constructs, which were low, medium, and high expression levels. The three plots represent the three BCD constructs, and the heights of the peaks align with the expected low, medium, and high expression levels. Although the intensity is quite low, the emission spectrum has a spike right where it should be for RFP. We believe that the low intensity of the fluorescence was due to an error in the dilution of the cell culture that we measured.
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