840:153g/2008

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840:153g: Recombinant DNA Te(a)chniques

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Contents

Foreword

"If you want truly to understand something, try to change it" (Kurt Lewin)

This page is the FINAL homepage of the course titled "840:153g". Current version: Fall 2008!

Course Overview

During this course, 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 designing your own experiments, self experimentation, record keeping, and discussion and reporting.

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 to 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 four (yes that's right: 4) throughout the course. Each team will design a new biological part or device, assemble it, prove it, and store it in the registry of biological standard parts. Your team may use all parts stored in the registries 2007 DNA distribution kit. The kit is available to you in form of DNA. Therefore, see these instructions how to use the kit.

Your Assignment

Like in the "International Genetically Engineered Machine" (iGEM) competition, you will work in teams to develop a new biological part, module, or device. Our teams are small, but I expect each team to develop something new - something that does not yet exist in the repository of biological standard parts. This is a very challenging task for the few sessions we have and not necessarily something all teams will be able to complete. Therefore, very good planning is required. The better your team plans the entire project from the beginning, the better your chances are to complete your project. Since good planning is very important, it will contribute 20% to your grade. Your team has to carefully describe your project on your project homepage and give a presentation during the third week of class.

It is up to each team to make a decision which new part to make. However, teams should carefully study the parts list to find something that's not already there. New parts can be as simple as a new protein coding device or regulatory region, a combination of existing parts, or something else you would like to do. However, we cannot synthesize DNA because that is too costly. Therefore, you have to work either with existing parts (from the 2007 repository) or use DNA that you can amplify by PCR from readily available resources (for example food plants). In addition to the particular part, module, or device your team wants to produce, each team needs to make sure that parts are biobricks compatible. This means your part must eventually be modified by site-directed mutagenesis to substitute for conflicting restriction sites (e.g. those used in the biobricks standard assembly). Finally, you (as a team) have to provide evidence that your part, module, or device is functioning in the way you predicted. Each team presents its results in another presentation after thanksgiving week. This presentation is again worth 20% of the grade.

How to Start

1) Select your part, module, or device

Your first team task is to select a project. Please take a look at the iGEM presentations for some ideas and an overview of what undergraduate teams achieved. Then, select a project your team wants to work on. Be curious and select something interesting. Don't be afraid to select a project that you eventually might not be able to complete. We will learn something from each project - regardless of how far you get with it. So please select a challenging and interesting project, something you really would like to do. Browse the parts in the registry to find out what is available to use and what you can build on. Then, make a list of what needs to be done. We will discuss all proposals in the third week.

2) Describe your project on the team homepage

You have to describe your project on the team homepage. The description should be very concise and contain enough details to understand the project in all its details. That includes:

  • desrciption of the product (part, module, device)
  • sources for cloning it (e.g. tissue, parts used)
  • steps required to make it
  • proof of function

Developing your ideas and figuring out what is involved is a complex task that involves all team members. You have to sit together and communicate your ideas. Brainstorm, make plans, assign work pieces to everyone, find information, discuss what you found, revise your concepts, look up protocols and describe your approach on your homepage. Familiarize yourself (everyone) with OpenWetWare and how to edit pages. Do not forget to apply for an account after (or during) the first class session. You will need it immediately. The sooner you start writing (even rough drafts, lists of ideas) the easier it will be for your team to keep track of your progress and find relevant information. And the more you write the better your grade will be. WRITE IN EVERY SESSION! The completeness and clarity of your strategy will be assessed and graded (20%) by the end of the third week. Your 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 or even start your project. Questions you cannot solve on your own (until your proposal is due at the end of week 2) should be listed at the end of your experimental strategy under the "Need to Know" section. This section can be quite large. A good selection of "need to know" questions is very helpful in developing your experimental approach. Ask yourself (and your team members): What information (about the part and the experimental procedures) do you need in order to produce and verify 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 revise and 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 consider this 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 lecturings on specific topics may be given occasionally and if needed.

Grading

Your team needs to develop a website at OWW where you describe your project (in form of a proposal). The first 10% of the grade will be given for selecting a part, module, device, developing an experimental outline to produce it, and completing the project proposal by the end of week 3. Your proposal should be complete by the end of week two to allow for a review. Each team will present its proposal in a 20 minute presentation in the last session of week 3. This presentation is worth another 10% of your grade.

A large and very important part of the class grade will be based on writing a detailed lab notebook. This personal 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! Everyone has to write her/his own notebook, but tasks should be equally shared among team members. It is absolutely necessary that everyone writes a good notebook and keeps it updated. The notebook will be worth 40% of your grade and graded 3 times during the semester (after 5, 10, 15 weeks). Notebook keeping is essential for your work and grade 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 or discuss progress within your team or with me.

Depending on the combined team notebooks, an online journal must be kept for each team (worth 15% of your grade). This journal has to be updated before the next lab session and summarize the teams achievements made during EACH session. I do not expect an exhaustive explanation or a simple repetition of your notes. All team members should sit together after class to SUMMARIZE the achievements, acknowledging every ones contribution. Don't forget to write an action plan (ToDo list) for the next lab. 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 every team members contribution.

Finally, I expect each team to develop their part and test it. If a group does not achieve it, the achievements as well as problems must be explained thoroughly in the online notebook. Evidence must be provided in the lab notebooks that sufficient efforts have been undertaken to achieve the goals mentioned in the proposal. After all, this is real science (none of the parts you attempt to develop have ever been generated) and there is no guarantee that any group will succeed (though chances are high). Therefore, keeping a good notebook is essential. The individual notebook will account for 40% of the grade. The online journal (project website) will account for another 15% of the grade. All team members will receive identical grades for the online journal (15%) and the two presentations (10% first presentation, 15% final presentation). However, everyone's individual contribution to the team progress will be weighed by three peer assessments during the semester.

I will assign another 10% of the grade based on my individual assessment of everyone's personal contribution. Here, I will consider ideas presented during class sessions, including preparation for class, discussion of experiments and results, timeliness, and teamwork capabilities. It is okay to have fun in the lab and there will be times where you have to wait for gels or PCRs to finish. However, use your time wisely. Don't waste it by discussing the latest movies or last weekends party. Time for experiments is scarce enough and each team will have a very tight schedule. This is not a class where you can just sit around and talk and let others to the work! Cell phones and other electronic devices (iPhones or MP3 players) are not allowed in the lab and will be confiscated if found.

Final Grade Composition:

  • 10% project proposal and experimental plan (due by end of week 2)
  • 10% team project presentation (due by end of week 3)
  • 40% lab notebook
  • 15% online journal (team website)
  • 10% individual contribution (ideas presented,preparation, performance, knowledge)
  • 15% final project presentation

Once again: four grades (50%) will be team grades for which all team mates will receive identical grades. The grades will be weighed by three peer evaluations from your team members. Two other grades (notebook and individual contribution) will be individual grades (50%). There are no make-ups possible in this class!!!

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 proposal. There is help available on how to use this WIKI in the left-handed navigation pane and in the top menu bar.

Tutorials

Check out this nice tutorial by Prof. Arking to get started in the lab.


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