840:153g/2008

Course Overview

This page is the PRELIMINARY homepage of the course titled "840:153g". Current version: Fall 2008! The page and course contents are still under development - please check back often!

You will be introduced to basic techniques for cloning and recombining DNA. The principles of gene cloning, DNA manipulation and analysis will be addressed from a practical view. This lab requires self experimentation, planning and designing experiments, critical thinking, and discussion.

Upon completion of the course, you will

1. understand how gene sequences are translated into protein in different organisms
2. conceptually understand how to manipulate DNA and create recombinant proteins or modify protein expression
3. be able to identify, analyze, and functionally recombine DNA molecules

By taking this course, you will learn how to isolate, analyze, and clone DNA. You will have a broad overview over the most common procedures used in a molecular biology laboratory. You will be able design and carry out simple experiments, document and explain your results. You will have demonstrated that you developed skills in finding and using genomic information stored in public repositories, analyze and summarize your findings, and logically organize and present your data.

You will work in teams of two throughout the course. Each team will (1) clone a hop (this is the name of the plant :-) gene, (2) fuse it to the green fluorescent protein (GFP), and (3) express the fusion protein in E. coli.

Your Assignment

This is were we start from: "You just started your new job in a plant genetic engineering lab. The laboratory manager tells you that the business management department came up with a new gene sequence that has recently been published. The lab manager was asked to produce a GFP-tagged fusion-protein of it in E. coli that can be used in transgenic plants to track down its subcellular location. He just remembers the accession number of the gene but forgot what it was. He asks you to look up the gene in the database and tell him what it codes for (select your favourite hop gene here). Then he asks you as the new research assistant to make a suggestion how to clone it and to advise a detailed experimental strategy to do so. This is why he hired you 6 weeks ago. Since he has to leave for a conference overseas he cannot provide you any assistance until he is back in two weeks. But he expects you to develop a thorough experimental plant until he returns. The only help he can provide is the assistance of the new undergraduate student who started working in the lab just this week. However, since she is new in the lab she has no clue how to do proceed. You two therefore sit together developing an experimental strategy. And you know: when the lab manager is back he expects your plan to be ready to go. And there are you just two weeks left to get this done."

How to Start

Your first team task is to select a gene from hop (Humulus lupulus) you want to clone. Select a structural gene from the flavonoid pathway, a flavonoid transporter, or a transcription factor. Each team works on a different gene and needs to provide the name of the gene and its accession number. After your selection has been approved by the instructor (by the end of the first week), you develop an experimental strategy to clone the fusion protein. Your strategy has to be explained in detail on your team members website (in OWW - see link "projects") under the "Project Description" section. The completeness and clarity of your strategy will be assessed and graded (10%) by the end of the second week.

Your team selects a gene from the NCBI database and clones the coding region only (no introns are allowed). You will develop your own strategy for cloning the gene, including a procedure for verification that you actually cloned the full coding region of the gene you selected. The approach must be written down in form of a research proposal on your project website and approved by the instructor before lab work can commence.

You almost certainly will identify a couple of gaps in your knowledge that have to be addressed in order to complete your project. The questions you cannot solve until your description is due (at the end of week 2) should be listed at the end of your experimental strategy under the "Need to Know" section. Ask yourself (and your team member): What information (about the gene and the experimental procedures) do you need to know in order to clone it? Prepare a list of issues you need to address! Keep in mind that the WIKI pages can be easily edited. So start writing as soon as possible. You can modify it later.

Please be aware that the first 2-3 weeks may indeed be spend developing your experimental plan rather than working in the lab. If you wish, you can view this part as the combined lecture part of the course.

Attendance Policy

Attendance at ALL laboratories is mandatory. Only religious observance, a death in the family, or serious illness/injury and a doctor\\\\\\\'s note are valid excuses. One unexcused absence from lab will result in failure of the course. Lecture and lab sessions are always combined. There will be no formal lectures but short lectures on specific topics may be given if needed.

Grading

Your team will need to develop a website at OWW in which you describe your project (in form of a proposal). The first 10% of the grade will be given for selecting a gene, developing an experimental outline, and completing the project description. This is due by the end of week two.

A large and very important part of the class grade will be based on the writing notebooks. A lab notebook is required that will be graded based on completeness, accuracy, and content. It should contain all experimental procedures carried out during each lab session (incl. observations, calculations, remarks, etc.), as well as description and discussion of results. Based on the results obtained, it should also contain a brief description of the steps you plan and materials you need for the next session. The notebook has to be written at the day of the lab - during the lab! Depending on these notes, an online journal must be kept. This journal has to be updated before the next lab session. No late entries (even 1 day) will be considered. Since each entry in the online journal receives a time stamp and the name of user who writes it, it very easy for me to monitor everyones contribution. Notebook keeping is considered an essential part of the work and therefore should be done during the lab sessions. There will often be lag times while experiments run. Use them for writing your lab notebook and updating the online journal (the computer room is right next door).

Finally, I expect each group to clone their fusion gene. If a group does not achieve it, the problems must be explained thoroughly in the online notebook and evidence must be provided from the lab notebook that sufficient efforts have been undertaken to clone the gene. After all, this is real science (none of the genes you will work with have ever been cloned) and there is no guarantee that any group will succeed (though chances are high). Therefore, keeping a good notebook is essential. The notebook will account for 40% of the grade (20% for online journal, 20% for the written notebook) of the grade and both partners receive the same grade for the notebook, weighed by a peer assessment. Both partners, however, should regularly switch responsibility to write notes.

I will assign 30% of the grade based on everybody's individual contribution. Here, I will consider contributions during class sessions, including preparation for class, discussion of experiments and results, timeliness, and teamwork. The remaining 20% will be given for the project presentation. One member of each team will give a midterm presentation (around mid October), the other member will give a final presentation (the week before finals). Both presentations should give a 10-minute overview of what has been done - and achieved - in the project. It will be essential to provide experimental data, describe and explain them carefully, and discuss them with classmates.

Final Grade Composition:

• 10% project description and experimental plan (due week 2)
• 20% lab notebook
• 20% online journal
• 30% individual contribution (preparation, performance, knowledge)
• 20% project presentation

Once again: the three first grades will be team grades for which both team mates will receive identical grades. The last two grades will be individual grades. There are no make-ups possible !!!

Getting Started with OWW

To get started with your online journal, you need to apply for an account at OWW (which is free). Please write down your username and password from your confirmation email. After you obtained your account details, please go to the Project Page and start developing your project. There is help available on how to use this WIKI in the left-handed navigation pane and in the top menu bar.