Regulatory

Part:BBa_K1225000

Designed by: James Nolan   Group: iGEM13_Purdue   (2013-09-16)


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

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]


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 CONDITIONS

Media 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

Notes: The growth curve on the right contains three curves: control BL21 cells, BL21 cells with just pSB1C3, and BL21 cells with pSB1C3 with the prtc* promoter inserted. We believe that the strain of producing the antibiotic resistance delayed the pSB1C3 and prtc* cultures from entering exponential phase at the same time the control BL21 cells did. Because the control cells entered the phase quicker, they also reached the stationary phase faster. Overall, we determined that the prtc* promoter has no significant effect on the growth of BL21 cells that would be problematic for future assays.



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 CONDITIONS

Media 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 graph shown, 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. The picture to the right shows the cells expressing RFP under blacklight.


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