Ms. Tara L. Southworth Profile

Tara L. Southworth

at Connecticut College

SPIE Involvement:

Author

Publications (1)

This will count as one of your downloads.

You will have access to both the presentation and article (if available).

DOWNLOAD NOW

This content is available for download via your institution's subscription. To access this item, please sign in to your personal account.

Email or Username Forgot your username?

Password Forgot your password?

Show

Keep me signed in

No SPIE account? Create an account

Proceedings Article | 14 June 2004 Paper

Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

Bruce Branchini, Tara Southworth, Neelum Khattak, Martha Murtiashaw, Sarah Fleet

Proceedings Volume 5329, (2004) https://doi.org/10.1117/12.530725

KEYWORDS: Bioluminescence, Proteins, In vivo imaging, Biological research, Magnesium, Acquisition tracking and pointing, Genetics, Manufacturing, Error analysis, Biomedical optics

Read Abstract +

Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

My Library

You currently do not have any folders to save your paper to! Create a new folder below.

Folder Name

Folder Description

View contact details

UPDATE YOUR PROFILE

Is this your profile? Update it now.

Sign into your SPIE.org account

Don’t have a profile and want one?

Create an account on SPIE.org

Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks. You are receiving this notice because your organization may not have SPIE eBooks access.*

*Shibboleth/Open Athens users─please sign in to access your institution's subscriptions.

To obtain this item, you may purchase the complete book in print or electronic format on SPIE.org.

ORGANIZATIONAL
Sign in with credentials provided by your organization.

Organizational Username

Organizational Password

Show/Hide Password

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

;

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available

Members:

Non-members: ADD TO CART

Keywords/Phrases

Search In:

Publication Years