The plasmid pCG001 expressing Ubp1 was kindly provided by Rohan Baker at the John Curtin School of Medical Research. This work was funded by March of Dimes Research Grant 1-FY2011, March of Dimes Basil O'Connor Starter Scholar Research Award #5-FY20 and NSF CAREER award 0746796.

1. Strain Engineering Workflow
<ol>
	<li>Insert sequences encoding (a) a membrane-localization sequence, (b) a fast-maturing fluorescent protein and (c) a protease recognition sequence N-terminal to and in frame with a protein of interest (e.g. a transcription factor). We used the membrane targeted Tsr-Venus reporter 2 and fused it to the protease recognition sequence Ubiquitin (Ub) to count the number of expressed bacteriophage &lambda; repressor CI protein molecules. Details on how we constructed ...

<ol>
	<li>Hensel, Z., Feng, H. D., Han, B., Hatem, C., Wang, J., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+expression+dynamics+of+transcription+factor+revealed+by+single-molecule+noise+analysis.">Stochastic expression dynamics of transcription factor revealed by single-molecule noise analysis.</a> Nat. Struct. Mol. Biol. 19, 797-802 (2012).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Gene+Expression+in+Live+Cells,+One+Protein+Molecule+at+a+Time.">Probing Gene Expression in Live Cells, One Protein Molecule at a Time.</a> Science. 311, 1600-1603 (2006).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Nat. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Tobias, J. W., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Functional+Analysis+of+the+Ubiquitin-specific+Protease+Gene+UBP1+of+Saccharomyces+cerevisiae.">Cloning and Functional Analysis of the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cerevisiae.</a> J. Biol. Chem. 266, 12021-12028 (1991).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , A2.2 (2001).</li><li>Xiao, J., Elf, J., Li, G., Yu, J., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gene+expression+in+living+cells+at+the+single-molecule+level.">Imaging gene expression in living cells at the single-molecule level.</a> Single Molecules: a laboratory manual. , 149-169 (2007).</li><li>Nagai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Stagaman, G., Forsyth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright-field+microscopy+of+semitransparent+objects.">Bright-field microscopy of semitransparent objects.</a> J. Opt. Soc. Am. A. 5, 648-659 (1988).</li><li>Tobias, J. W., Shrader, T. E., Rocap, G., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+in+bacteria.">The N-end rule in bacteria.</a> Science. 254, 1374-137 (1991).</li></ol>

The CoTrAC method can be generalized to measure the production of other proteins where conventional N- or C-terminal fluorescent protein fusions may disrupt protein activity. The CoTrAC strategy has three unique advantages over current methods. First, co-translational fusion ensures that one molecule of the fluorescent reporter is produced for each molecule of the protein of interest, allowing accurate counting of protein production in real time. Second, the membrane-targeted reporter Tsr-Venus enables single-molecule de...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Agarose (Low melting temperature) </td>
 <td>Lonza</td>
 <td>50100</td>
 <td></td>
 </tr>
 <tr>
 <td>Milli-Q H2O</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>5&times;M9 salts</td>
 <td></td>
 <td></td>
 <td>Following recipe described in 9</td>
 </tr>
 <tr>
 <td>20% glucose</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>50&times;MEM amino acid solution</td>
 <td>Invitrogen</td>
 <td>11130-051</td>
 <td></td>
 </tr>
 <tr>
 <td>Temperature-Controlled Growth Chamber 
 Stage adaptor</td>
 <td>Bioptechs</td>
 <td>FCS-2</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective Heater</td>
 <td>Bioptechs</td>
 <td>Model depends on microscope objective</td>
 <td></td>
 </tr>
 <tr>
 <td>Microaqueduct Slide</td>
 <td>Bioptechs</td>
 <td>130119-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro cover glasses</td>
 <td>VWR</td>
 <td>40CIR-1</td>
 <td>Can be difficult to source; also available from Bioptechs</td>
 </tr>
 <tr>
 <td>Cover glass/slide gasket</td>
 <td>Bioptechs</td>
 <td>FCS2 0.75 mm</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorescence Microscope</td>
 <td>Various</td>
 <td>Example setup: Coherent Innova 308C Argon-ion laser, Olympus IX-81 microscope, Olympus PlanApo 100X NA 1.45 objective, Metamorph software</td>
 <td>Must have laser excitation, automated xyz stage, automation software capable of scripted imaging and autofocus, optics capable of resolving single fluorescent proteins </td>
 </tr>
 <tr>
 <td>EM-CCD Camera</td>
 <td>Various</td>
 <td>Example setup: Andor Ixon DU-898</td>
 <td>Must have sufficiently low noise to detect single fluorescent proteins above background</td>
 </tr>
 </tbody>
</table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Typical results from a CoTrAC experiment tracking the production of the &lambda; repressor CI are shown in Figures 4 and 5. In this experiment, 12 colonies were imaged at 5 min intervals. At each time point, the colony was first autofocused and centralized within the imaging region. Next, the centered/focused position was stored and a brightfield image was acquired. The stage was then translocated by ~0.5 &mu;m along the z-axis to move from the brightfield focal plane to the plane bisect...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

single-molecule-imaging-gene-regulation-vivo-using-cotranslational

Research

JoVE Journal

Biology

9.8K visualizações.  Johns Hopkins University School of Medicine. Descreve-se um método de fluorescência microscopia, Activation Co-translacional pela clivagem (CoTrAC), a imagem a produção de moléculas de proteína de células vivas com uma única molécula de precisão, sem perturbar a funcionalidade da proteína. Este método tem sido utilizado para seguir as dinâmicas de expressão estocásticos de um factor de transcrição, o repressor CI λ 1.