Difference between revisions of "Part:BBa K1225000"
 
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<partinfo>BBa_K1225000 short</partinfo>
 
  
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<b>PART DESCRIPTION:</b>Constitutive  promoter
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<html>
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<b>PART DESCRIPTION:</b> Constitutive  promoter
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<br><p>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.</p></br>
  
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<p>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.</p>
 
  
 
<HR>
 
<HR>
  
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<h1>Contact Information</h1>
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<b><h3>Contact Information</h3></b>
  
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<ul style="list-style: none;">
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<br>
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<li>Author(s): James Nolan and Chris Thompson</li>
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<li>Team: Purdue University 2013</li>
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<p><u>Author(s):</u> James Nolan and Chris Thompson</p>
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<li>Data Collection: Amanda Shanley, Chris Thompson, and James Nolan</li>
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<p><u>Team:</u> Purdue University 2013</p>
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<li>Affiliation: Purdue University (Bindley Bioscience Center)</li>
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<p><u>Data Collection:</u> Amanda Shanley, Nidhi Menon, Chris Thompson, and James Nolan</p>
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<li>Contact: nolan6@purdue.edu</li>
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<p><u>Affiliation:</u> Purdue University (Bindley Bioscience Center)</p>
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<li>Related Parts: None</li>
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<p><u>Contact:</u> nolan6@purdue.edu</p>
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<li>Date: 09/27/13</li>
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<p><u>Related Parts:</u> None</p>
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</ul>
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<p><u>Date:</u> 09/27/13</p>
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</br>
  
 
<HR>
 
<HR>
  
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</html>
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<b><h3>Standard Design Information</h3></b>
  
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<span class='h3bb'>Sequence and Features</span>
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</html>
 
<partinfo>BBa_K1225000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1225000 SequenceAndFeatures</partinfo>
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<html>
  
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<br>
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<p><u>Chassis:</u> E.coli</p>
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<p><u>Strain:</u> BL21</p>
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<p><u>Device Name:</u> BBa_K1225000</p>
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<p><u>Device Type:</u> Promoter</p>
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<p><u>Safety Level:</u> Risk Group 1</p>
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<p><u>Assembly:</u> Golden Gate Assembly</p>
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<p><u>Protocol:</u> Freiburg Golden Gate Protocol</p>
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<p><u>Scars:</u> None</p>
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<p><u>Insertion:</u> Plasmid</p>
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<p><u>Vector:</u> pSB1C3</p>
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</br>
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<HR>
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<b><h3>Growth Rate Assay</h3></b>
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<br>
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<b>BASIC INFORMATION</b>
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<p><u>Purpose:</u> To assess what effect, if any, our genetic parts have on the growth rate of E.coli.</p>
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<p><u>Chassis:</u> E.coli</p>
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<div align="right" z-index:100><img src="https://static.igem.org/mediawiki/2013/5/51/Growth_Curve.png" width="500" height="300" align="right"></div>
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<p><u>Strain:</u> BL21</p>
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<p><u>Protocols:</u> Purdue iGEM Growth Curve Protocol</p>
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<p><u>Date:</u> 09/25/13</p>
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</br>
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<b>GROWTH CONDITIONS</b>
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<p><u>Media Type:</u> Luria Broth (LB)</p>
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<p><u>Vessel:</u> 10mL Culture Tube</p>
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<p><u>Volume:</u> 5mL</p>
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<p><u>Incubation:</u> 37 C, 250 rpm</p>
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<br>
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<b>MEASUREMENT INFORMATION</b>
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<p><u>Data Type:</u> Growth Curve (OD vs Time)</p>
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<p><u>Location:</u> Bindley Bioscience Center</p>
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<p><u>Machine Name:</u> N/A</p>
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<p><u>Time Interval:</u> 30min</p>
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<p><u>Total Time:</u> 270min</p>
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<p><u>Notes:</u> 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.</p>
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</br>
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<hr>
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<b><h3>Proof of Functionality</h3></b>
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<br>
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<b>BASIC INFORMATION</b>
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<p><u>Purpose:</u> To qualitatively and quantitatively prove that our promoter initiates transcription by placing mRFP after it.</p>
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<p><u>Chassis:</u> E.coli</p>
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<div align="right" z-index:100><img src="https://static.igem.org/mediawiki/2013/1/10/RFPIntensity.png" width="500" height="300" align="right"></div>
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<p><u>Strain:</u> BL21</p>
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<p><u>Protocols:</u> None</p>
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<p><u>Date:</u> 09/26/13</p>
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</br>
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<b>GROWTH CONDITIONS</b>
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<p><u>Media Type:</u> Luria Broth (LB)</p>
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<p><u>Vessel:</u> 10mL Culture Tube</p>
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<p><u>Volume:</u> 5mL</p>
 +
<p><u>Incubation:</u>37 C, 250 rpm</p>
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<br>
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<b>MEASUREMENT INFORMATION</b>
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<p><u>Data Type:</u> Fluorescence Intensity vs Wavelength</p>
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<p><u>Location:</u> Bindley Bioscience Center</p>
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<div align="right" z-index:100><img src="https://static.igem.org/mediawiki/2013/c/ce/RFPPicture2.png" width="300" height="200" align="right"></div>
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<p><u>Machine Name:</u> Cary Eclipse<p>
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<p><u>Excitation:</u> 584nm</p>
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<p><u>Emission:</u> 600nm-620nm</p>
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</br>
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<p><u>Notes:</u> 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.</p>
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</html>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 03:43, 26 November 2014


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, Nidhi Menon, 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.