|
Welcome
"Teach with SSB" is a laboratory teaching module with various experiments designed around the activities of single strand DNA binding (SSB) proteins. The experiments are ideal for Biochemistry/Molecular Biology and Biophysics classes. The modules are developed by Dr. Edwin Antony at Marquette University. Please feel free to adapt the entire course material or you can select individual experiments and integrate them into existing courses. Please contact us if you require course material, plasmids for protein expression or E. coli overexpression strains. We would be delighted to hear from you if you have ideas to improve the experiments or to add new experiments to our library. Thanks for your interest! Advantages of using the SSB protein for teaching Rapid, Column-Free Purification: The E. coli SSB protein provides a stable, inexpensive and experimentally versatile tool around which the curricula of graduate, undergraduate and high school science courses can be formulated. For example, the SSB protein can be purified without the use of any chromatographic steps and with simple precipitation using Polymin P and ammonium sulfate (Figure 1A). Long-term Stability: The purified protein can be stably stored for several years at 4 °C in solution or as an ammonium sulfate pellet. It is also a stable protein without any degradation (Figure 1B) or loss in DNA binding activity (Figure 1C) even after remaining at room temperature for three weeks. |
Student Cover Art
These images were generated by the students in the BIOL4102 class (Fall 2015). They have conveyed their scientific findings using a cohesive artistic rendering, suitable for public dissemination. |
Figure 1. The stability of SSB renders it a suitable experimental tool for teaching applications. A. SDS-PAGE showing samples from an inexpensive column-free strategy to purify SSB proteins. Post lysis, SSB is subject to simple polymin-P and ammonium sulfate precipitation which yields > 95 % pure SSB protein. B. A SDS-PAGE gel showing the stability of the SSB protein after leaving it at room temperature (RT) for three weeks. C. DNA binding experiment done with SSB protein stored at -20 ˚C or after three weeks at RT. Both samples show ssDNA binding activity with each tetramer stoichiometrically binding to one (dT)70 ssDNA molecule.
Highly Quantitative and Observable Signals: SSB binds DNA rapidly with very high affinity and to unity. This means molar equivalents of SSB will bind to molar equivalents of single stranded DNA (with a binding site size of 70 nt). You can teach a broad variety of scientific principles: from simple techniques such as the importance of pipetting and concentration measurements, to more sophisticated experiments such as crystallography, ITC, AUC and single molecule measurements.
Mutiple Assays: EMSA, Fluorescence, ITC, Stop flow, protein-protein interactions, Western Blots, Standard Molecular Biology, X-ray crystallography, Filter Binding, etc. If you have an experiment/technique in mind, you should be able to choose an activity of SSB to investigate. If its a graduate lab with high tech gear (single molecule TIRF, EM, CD), undergraduate lab with medium tech gear (EMSA, SDS-PAGE, cloning) or a high school lab with low tech gear (agar plates and colony counting) SSB is amenable to all these approaches. The advantage is often a large quantifiable signal for all such experiments. SSB DNA complex crystals can be formed within a couple of days and diffraction datasets can be collected in house to 2.4 Angstroms.
Bridge the In vitro - In vivo Ideas Effortlessly: Teaching students the link between biochemical/biophysical experiments and what they mean to life in general is one of the hardest parts of an experimental course. With SSB proteins, one can test in vivo complementation of various SSB mutants through simple E.coli transformation and a bumping experiment where loss of a plasmid can be tracked through screening for antibiotic resistance.
Mutiple Assays: EMSA, Fluorescence, ITC, Stop flow, protein-protein interactions, Western Blots, Standard Molecular Biology, X-ray crystallography, Filter Binding, etc. If you have an experiment/technique in mind, you should be able to choose an activity of SSB to investigate. If its a graduate lab with high tech gear (single molecule TIRF, EM, CD), undergraduate lab with medium tech gear (EMSA, SDS-PAGE, cloning) or a high school lab with low tech gear (agar plates and colony counting) SSB is amenable to all these approaches. The advantage is often a large quantifiable signal for all such experiments. SSB DNA complex crystals can be formed within a couple of days and diffraction datasets can be collected in house to 2.4 Angstroms.
Bridge the In vitro - In vivo Ideas Effortlessly: Teaching students the link between biochemical/biophysical experiments and what they mean to life in general is one of the hardest parts of an experimental course. With SSB proteins, one can test in vivo complementation of various SSB mutants through simple E.coli transformation and a bumping experiment where loss of a plasmid can be tracked through screening for antibiotic resistance.
|
BIOL 4102 at Marquette University
Fall 2016 is the second year that we are using SSB proteins as our focus of investigation. Under the 'Experiments', you will find the course material, notes and syllabus. Under the 'Blog' Tab, you will student discussion of the data they have obtained. It also provides a student view of learning objectives and their take on this teaching journey. In this course, students are cloning out SSB genes from various pathogenic bacteria, purifying the protein and investigating their biochemical and biophysical properties. |
Sam and Kaitlin, students in the Fall 2015 BIOL 4102 class, perform a SSB-DNA binding experiment using our Stop Flow instrument.
|
Ally and Hanna (BIOL 4102 students) try out the incubator to grow their cells.
|
Tryg and Hanna (BIOL 4102 students) get a tour of the quench flow setup
|
