Conditions of Use
This lab manual is designed for graduate students but it seems it could easily be designed to work as a CURE at a university with necessary resources. This laboratory experiment seems to be designed as a semester long unit. The project does a... read more
This lab manual is designed for graduate students but it seems it could easily be designed to work as a CURE at a university with necessary resources. This laboratory experiment seems to be designed as a semester long unit. The project does a reasonably good job at presenting the central dogma in a different light. This text is likely to refer you to other sources for descriptions. This left gaps in the comprehensiveness because it seems like this could be summarized and cited. Often the reader was left with incomplete citations at the end of each module. I would hate for a student to have to buy an excess of other resources to understand the content.
The descriptions of the experimentation was accurate as were the concepts.
This content is exciting and an interesting way to look at enzyme kinetics. Enzyme kinetics will always be relevant! Additionally, I think the use of the Genetic Code Expansion amino acids is a creative and novel way of addressing protein structure and function. I am not a biochemist, so I admit I may be incorrect, but haven’t most biochemists moved away from the Michaelis-Menten approach to enzyme kinetics in favor of the Lineweaver Burk plots? The calculation time is the comparable, but you end up with a more precise Vmax and Km. The longevity of the experimentation was well suited for a semester long lab. The lab however does require the use of pyMol for the protein modeling portion of the lab. As someone who often manipulates pdb files and protein models, I struggled with this program and did not find the provided instructions intuitive.
The introductory material that was presented was very clear and easy to follow. Reading through, I could very easily identify the goals and controls necessary for each assignment. This experiment as a resource still seems specific to the institution in which it was designed. I was unfamiliar with GCE prior to this review, and was surprised the term was not defined in the introductory material. Later in the experimental sections, the text gets heavy with Socratic questioning that would be helpful to a student that is not struggling with understanding the concepts, though I struggled to see how it would help a student struggling. At some points in the text, it seems as though more than half of the text is devoted to these questions
This text follows consistent presentation with concepts, methods, and objectives clearly delineated.
This lab manual naturally is divided into it weekly objectives. The amount of background material provided was sufficient but not overwhelming for a student in one week sections.
The presentation of the material and the experimentation was logical. Presentation of background information was excellent in the beginning of the lab manual though later in the manual I struggled with so many references to other texts. The concepts alluded to in other texts did not seem to be negligible for proper understanding of the experiment.
I did not encounter any issues when navigating the E-book.
The text contains no grammatical errors. On rare occasions, figures are presented but not referenced in text. Figure legends were occasionally incomplete.
The text is not culturally insensitive or offensive.
The use of GCE is a timely and students who learn from this technique will benefit from the course.
This Lab Manual is for graduate students and may also be used as an experimental framework for independent research. Readers/students must know the principles of molecular biology and biochemistry, and also be familiar with the terminology and... read more
This Lab Manual is for graduate students and may also be used as an experimental framework for independent research. Readers/students must know the principles of molecular biology and biochemistry, and also be familiar with the terminology and techniques of bacterial cell culture, molecular cloning, targeted mutagenesis, protein purification, and enzymatic assay. Though it is clear from the title (BB 495) that the course is highly specialized, it would be appropriate to mention these prerequisites at the beginning, right before “Background”. Looking from the students’ perspective, this book could be used rather as an auxiliary laboratory guide for their existing courses or lab works. The chapters of this manual provide the general concepts which are fully comprehensive for prepared students and describe the purpose of the experiments. There are only general outlines of the experiments, though some small but very useful tips are given here and there along with the lists of reagents and buffers’ compositions. The section “Deep Thoughts” at the end of a chapter/experiment is particularly useful for students – it urges to repeat and understand the material and promotes critical thinking. In many cases, authors refer readers to the other widely used lab manuals which I consider as a substitution for a glossary or indexes. Authors also suggest students use intensively PyMOL and PDB and analyze the existing literature. The enzymatic assay is just generally outlined, the experimental details are absent. I find this lab-book comprehensive and helpful for Teaching Professors and Lab Instructors.
I find the concepts and experimental descriptions being accurate. I would just disagree with the statement given in the “Deep Thoughts on Kinetics and Assays”: “Interpreting your results in a meaningful way may be one of the most challenging parts of your experiments.” The most challenging part of an enzymatic assay is to design the series of experiments in such a way that it produces results what are relatively easy to interpret. In most cases it is necessary to run two or more experiments under the same conditions but only one variable changed.
The content is relevant. The methods described are modern and up to date.
The description of purpose and process of experiments is mostly clear. It is suggested that readers must be familiar with the terminology used in the text. The text would be more clear with appropriate pictures; there are only three pictures used in this manual.
The structure of the text and terminology are consistent.
The logical sequence of the experiments is described at the beginning. The titles of the chapters are going in certain order - first goes "Concepts..." and then "Methods..." for the same experimental part. But the given time frame for the experiments and research is confusing. It is unclear how the big portion of the experiment can be "Objective for week 1". In the process of reading I realized that appropriately transformed bacteria is simply given to students. The process of overexpression and purification of the recombinant or ncAA-protein is described well, but the experimental part for the kinetics is simply left out.
The organization of the text is logical.
The text does not have an interface problems; can be read in different formats. All the chapters in the content are clickable, it is easy to navigate. All the links provided in the text are working.
I did not notice any grammatical error in the text.
The text is completely neutral; it is not culturally insensitive or offensive in any way.
I find this lab-book comprehensive and helpful for Teaching Professors and Lab Instructors.
Table of Contents
- 1. Background
- 2. Overview of Laboratory Responsibilities Tentative Schedule
- 3. Objectives for Week 1
- 4. Concepts for Choosing and Expressing Mutants
- 5. Objectives for Week 2
- 6. Concepts for Protein Expression
- 7. Methods for Protein Expression
- 8. Concepts for SDS-PAGE
- 9. Methods for SDS-PAGE
- 10. Objectives for Week 3
- 11. Concepts for Protein Purification and Desalting
- 12. Methods for Protein Purification and Desalting
- 13. Microfluidizer Lysis Method
- 14. Objectives for Week 4
- 15. Methods for Protein Standard Curves and SDS-PAGE
- 16. Objectives for Weeks 5-9
- 17. Concepts for Enzyme Assays
- 18. Methods for Enzyme Assays
About the Book
Chemical Biology & Biochemistry Laboratory Using Genetic Code Expansion Manual
About the Contributors
Ryan Mehl, Oregon State University
Kari van Zee, Oregon State University
Kelsey Kean, Oregon State University