Biomolecular Breadboards:DNA parts

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m
m (Negatively Autoregulated Gene)
(12 intermediate revisions not shown.)
Line 1: Line 1:
{{Template:Biomolecular Breadboards}}
{{Template:Biomolecular Breadboards}}
 +
{{righttoc}}
 +
This page contains a description of the DNA parts used for the biomolecular breadboards.  These parts will be submitted to [http://addgene.org Addgene] (eventually).
-
Under Construction
+
== Control Plasmid: pBEST-OR2-OR1-Pr-UTR1-deGFP-T500==
 +
 
 +
This section contains a description of the deGFP control plasmid for the cell-free expression breadboard.
 +
 
 +
{|
 +
|- valign=top
 +
| [[Image:pBEST_OR2_OR1_Pr_UTR-deGFP-T500.png]]
 +
|
 +
* Control sequence: [[Media:pBEST_OR2_OR1_Pr_UTR-deGFP-T500.gb|pBEST_OR2_OR1_Pr_UTR-deGFP-T500.gb]] (GenBank file)
 +
|}
 +
 
 +
=== Plasmid description ===
 +
{| border=1 width=100% cellpadding=4
 +
|- valign=top
 +
| P70
 +
| Lambda phage promoter OR2-OR1-Pr specific to E. coli σ70
 +
| GenBank: J02459.1
 +
|- valign=top
 +
| UTR1
 +
| The untranslated region containing the T7 g10 leader sequence for highly efficient translation initiation
 +
| GenBank: M35614.1
 +
|- valign=top
 +
| deGFP
 +
| The enhanced green fluorescent protein truncated and modified in N- and C- terminal
 +
| Shin, J., and Noireaux, V. (2010) Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70, J Biol Eng 4, 8.
 +
|- valign=top
 +
| T500
 +
| Transcription terminator for E. coli RNA polymerase
 +
| Larson, M. H., Greenleaf, W. J., Landick, R., and Block, S. M. (2008) Applied force reveals mechanistic and energetic details of transcription termination, Cell 132, 971-982.
 +
|}
 +
 
 +
=== Preliminary data ===
 +
 
 +
Using pBEST-OR2-OR1-Pr-UTR1-deGFP-T500, a plasmid enhanced for GFP expression, the biomolecular breadboard is able to express mass at equal concentrations to comparable bacteriophage in-vitro systems (J. Shin and V. Noireaux, 2010).
 +
 
 +
Expression of plasmids can be optimized by concentration.
 +
 
 +
{|
 +
|-
 +
|
 +
[[Image:plasmid_sat.png|400px]]
 +
| valign=top |
 +
* Raw data file: [[Media:plasmid_deGFP-070812.xls|plasmid_deGFP-070812.xls]] (Excel spreadsheet)
 +
* Information about protocol on "Outline" sheet; plotted data is on the sheet "plotted-data"
 +
<hr>
 +
* Control plasmid file: [[Media:pBEST_OR2_OR1_Pr_UTR-deGFP-T500.gb|pBEST_OR2_OR1_Pr_UTR-deGFP-T500.gb]] (GenBank format)
 +
|}
 +
 
 +
 
 +
'''Figure 1. eGFP expression as a function of plasmid DNA template.''' Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is varied by concentration.
 +
[[Image:pTet-TetR-deGFP (fusion).png]]
 +
 
 +
== Bistable toggle switch: 4 plasmid circuit==
 +
{|
 +
|-
 +
| align="center" | pTet-deGFP (pBEST-pLTet01-UTR1-deGFP-T500)
 +
[[Image:pTet_deGFP.gif]]
 +
| align="center" |
 +
pTet-LacI (pBEST-pLTet01-UTR1-LacI-T500)
 +
[[Image:pTet_LacI.gif]]
 +
|}
 +
 
 +
{|
 +
|-
 +
| align="center" | pLac-deCFP (pBEST-pLLac01-UTR1-deCFP-T500)
 +
[[Image:pLac-deCFP.gif]]
 +
| align="center" |
 +
pLac-TetR (pBEST-pLLac01-UTR1-TetR-T500)
 +
[[Image:pLac-TetR.gif]]
 +
|}
 +
 
 +
==Negatively autoregulated gene==
 +
 
 +
pTet-TetR-deGFP (fusion) (pBEST-pLTet01-UTR1-TetR-linker-deGFP-T500)
 +
 
 +
[[Image:pTet-TetR-deGFP (fusion).gif]]

Revision as of 02:10, 22 November 2012

Home Protocols DNA parts Preliminary Data Models More Info

Contents

This page contains a description of the DNA parts used for the biomolecular breadboards. These parts will be submitted to Addgene (eventually).

Control Plasmid: pBEST-OR2-OR1-Pr-UTR1-deGFP-T500

This section contains a description of the deGFP control plasmid for the cell-free expression breadboard.

Image:pBEST_OR2_OR1_Pr_UTR-deGFP-T500.png

Plasmid description

P70 Lambda phage promoter OR2-OR1-Pr specific to E. coli σ70 GenBank: J02459.1
UTR1 The untranslated region containing the T7 g10 leader sequence for highly efficient translation initiation GenBank: M35614.1
deGFP The enhanced green fluorescent protein truncated and modified in N- and C- terminal Shin, J., and Noireaux, V. (2010) Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70, J Biol Eng 4, 8.
T500 Transcription terminator for E. coli RNA polymerase Larson, M. H., Greenleaf, W. J., Landick, R., and Block, S. M. (2008) Applied force reveals mechanistic and energetic details of transcription termination, Cell 132, 971-982.

Preliminary data

Using pBEST-OR2-OR1-Pr-UTR1-deGFP-T500, a plasmid enhanced for GFP expression, the biomolecular breadboard is able to express mass at equal concentrations to comparable bacteriophage in-vitro systems (J. Shin and V. Noireaux, 2010).

Expression of plasmids can be optimized by concentration.

  • Raw data file: plasmid_deGFP-070812.xls (Excel spreadsheet)
  • Information about protocol on "Outline" sheet; plotted data is on the sheet "plotted-data"


Figure 1. eGFP expression as a function of plasmid DNA template. Plasmid DNA pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is varied by concentration. Image:PTet-TetR-deGFP (fusion).png

Bistable toggle switch: 4 plasmid circuit

pTet-deGFP (pBEST-pLTet01-UTR1-deGFP-T500)

Image:pTet_deGFP.gif

pTet-LacI (pBEST-pLTet01-UTR1-LacI-T500) Image:pTet_LacI.gif

pLac-deCFP (pBEST-pLLac01-UTR1-deCFP-T500)

Image:pLac-deCFP.gif

pLac-TetR (pBEST-pLLac01-UTR1-TetR-T500) Image:pLac-TetR.gif

Negatively autoregulated gene

pTet-TetR-deGFP (fusion) (pBEST-pLTet01-UTR1-TetR-linker-deGFP-T500)

Image:pTet-TetR-deGFP (fusion).gif

Personal tools