Checkmate: A Rapid Yeast Mating Type Detector Rose-Hulman Institute - - PowerPoint PPT Presentation

Checkmate: A Rapid Yeast Mating Type Detector

Rose-Hulman Institute of Technology

Ben Deschaine, Robert French, Alex Krug, Adam Nighswander, Kristen Schackmann, and Devon Trumbauer

Purpose

Goal:

To develop a system that significantly decreases the time required to determine the mating type of yeast (Saccharomyces cerevisiae) haploids

Application:

To facilitate basic research and commercial applications that utilize yeast

http://commons.wikimedia.org/wiki/File%3AYeast_lifecycle.svg

Yeast Life Cycle

Current Mating Type Test

Checkmate Mating Type Test

Benefits of Checkmate Method

• No need for complementary auxotrophic mutations

for diploid selection

• Significantly decreases time required for mating

test

Select diploids (~36 hrs) Mate haploids (~4 hrs) Checkmate! (~4 hrs) Traditional (~40 hrs) Start

Ajo-Franklin et al., Rational Design of Memory in Eukaryotic Cells

Construct Design

• Bifunctional promoter is bound by Ste12 and LexA
• Fluorescent domains act as reporters
• LexA DNA-binding domain and VP64 activator

together act as a transcription factor

• Nuclear localization sequence sends the

heteroprotein into the nucleus

Molecular Animation of Circuit

http://www.youtube.com/watch?v=i1yUjHN9Fyg

Project Obstacles

• After considerable effort, we determined that the

first vendor’s protocols were not amenable to producing the construct we desired

• After placing an order with a second vendor, we found

that the construct requested was much more difficult to synthesize than anticipated, so it was not received until mid-September

Collaboration with NTNU

Promoter Characterization

• BBa_K822000: lactate

promoter from E. coli

• Fused to BBa_E0422: cyan

fluorescent protein

• Constructs were sequenced

and verified

• Tested 100 mM lithium

lactate with controls

• Results were negative

Kofahl and Klipp, Modeling the dynamics

• f the yeast pheromone pathway

Differential Models

50 100 150 200 20 40 60 80 100 120 Protein Signal

Numerical Analysis: Gamma = 100

20 40 60 80 100 120 20 40 60 80 100 120 Protein Signal

Numerical Analysis: Epsilon = 1000

Outreach

• Aimed at two different age groups
• Long-lasting impact
• Affects the community as well as a larger

audience

Exploring DNA at the Design Table

Hands-on Experience at the Lab Bench

BioPioneer: Synthetic Biology edition is aimed at teaching players about synthetic biology as they play it. Players manage a laboratory to create a DNA strand that codes for the solution to

• ne of the six major world problems:

Malaria, Environmental Clean-Up, World Hunger, Alternative Energies, HIV Pandemic, and Malnutrition. Back Front Back Front

Players accomplish this through the use of lab personnel and 3 main strategies:

Lab personnel allow the player to use actions to work the equipment or write a paper. They also count towards the player’s knowledge pool. Each paper is different, and some can add to the player’s knowledge pool. Others have special abilities that optimize equipment or give the player a new ability. Equipment manipulates the DNA sequence to help it match the random sequence. Each lab has a special technique used in synthetic biology that helps them create their DNA sequence.

High School iGEM Competition

Questions?