Team:BIOTEC Dresden/RFC Novel Normalization Standard using Fluorescence

From 2010.igem.org

BBF RFC #: Novel Normalization Standard using Fluorescence

Syed Habib Tahir Bukhari, Ashwini Rahul Akkineni, Adithya Ananth, Victor Gordeev, Svea Grieb, Sarah Mansour, Mareike Roth, Charanya Sampathkumar, Lucas Schirmer, Jonathan Tam

27th October 2010

1. Purpose

The Biotec_Dresden Team 2010 developed an approach where two fluorescent proteins are simultaneously expressed. The fact that one reporter, in our case RFP, is constitutively expressed allows to monitor cell growth. Secondly, an inducible promoter drives the expression of the second reporter, YFP in the case of the part tested. The constitutively expressed reporter protein (R1) serves as normalization factor for the inducible reporter (R2) by simple division.

2. Relation to other BBF RFCs

BioBrick Assembly Standards
  • 1.12 BBF RFC 11: BioBrick™ assembly standard modifications
  • 1.15 BBF RFC14: Protein domain fusions in BB-2 assembly
  • 1.26 BBF RFC 25: Fusion Protein (Freiburg) BioBrick™ assembly standard
  • 1.27 BBF RFC 26: In-Fusion BioBrick™ Assembly
BioBrick Determination of Promoter Activity
  • 1.20 BBF RFC 19: Measuring the Activity of BioBrick™ Promoters Using an In Vivo Reference Standard

3. Copyright Notice

Copyright (C) The BioBricks Foundation (20##). All Rights Reserved.

4. Background

Performing kinetic measurements involving fluorescence in living cultures like bacteria requires the monitoring of growth generally performed using optical density (OD) measurements. These help to normalize the obtained fluorescent data and gain the actual values of fluorescence containing information about the actual cell number contributing to the overall fluorescence. To date this procedure is routinely followed.

5. Novel Approach

Instead of using OD measurements for normalization of fluorescent data R1 is introduced to the analyzed DNA construct containing inducible R2. Tests showed that this normalization standard is comparable and even more advanced than the routinely performed OD procedure.

6. Standard Procedure

  • Assemble R1 with the target construct using any of the mentioned BioBrick standard assemblies (Chapter 2) resulting in the following properties
  • R1 MUST be constitutively expressed
  • Both fluorescent reporters MUST NOT have an overlapping emission spectra
  • Both fluorescent reporters SHOULD NOT have an overlapping emission spectra
  • R2 SHOULD only be expressed upon desired induction
  • The complete construct MUST NOT include a negative feedback loop
  • R2 MUST NOT express a pigment
  • Promoter activity SHOULD be defined
  • 7. Author’s Contact Information

    Emali: User@domain
    Syed Habib Tahir Bukhari: habib-tahir.syed-bukhari biotec.tu-dresden.de
    Ashwini Rahul Akkineni: ashwini-rahul.akkineni biotec.tu-dresden.de
    Adithya Ananth: adithya.ananth biotec.tu-dresden.de
    Victor Gordeev: victor_gordeev yahoo.com
    Svea Grieb: maj-svea.grieb biotec.tu-dresden.de
    Sarah Mansour: sarah.mansour biotec.tu-dresden.de
    Mareike Roth: Mareike.Roth gmx.de
    Charanya Sampathkumar: charanya.sampathkumar biotec.tu-dresden.de
    Lucas Schirmer: lucas.schirmer yahoo.de Jonathan Tam: jywtam gmali.com

    References

    http://2010.igem.org/Team:BIOTEC_Dresden/Results

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