840:153g/2009/fall

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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 course homepage of the "Recombinant DNA Techniques" (840:153g) of the University of Northern Iowa. Current version: Fall 2009! You may want to watch this really good video from Drew Endy before (or after) you read on. Please also read his groundbreaking article about Foundations for Engineering Biology.

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, discussion, writing and reporting. Each session requires good preparation by the student for the laboratory tasks at hand.

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 functionally recombined DNA fragments
  3. be able to practically identify, analyze, and functionally recombine DNA molecules

You will have a broad overview over the most common procedures used in a molecular biology laboratory. This includes isolating DNA from different organisms, amplifying DNA through PCR, ligating DNA fragments together, tranforming E. coli with recombined plasmids, maintaining and growing bacterial stocks harboring recombined DNA fragments, analyzing DNA fragments on agarose gels, cutting DNA fragments with restriction enzymes, identifying DNA sequences in computer databases, designing oligonucleotides for PCR amplifications, using BLAST as a tool search for specific DNA fragments in online databases. 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 two 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 iGEM 09 DNA distribution. The kit is available to you in form of DNA as described in the above link. Please make yourself familiar with how to properly use the '09 DNA distribution. READ THE INSTRUCTIONS.

Your Assignment

Like in the "International Genetically Engineered Machine" (iGEM) competition, you will work in a small team 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 accomplish. But please do not worry about the outcomes. Since they are pretty unpredictable and depend on a large extend on the project you select to work on, the results will not affect your grade. However, in order to most likely succeed, very good planning is required. The better your team plans the entire project from the beginning, the better your chances are being able to complete your project. The planning is very important and it will contribute 10% 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. Based on the quality of your presentation, the understanding you show, and completeness or your experimental design, you now may (or not) start your bench work.

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 with existing parts from any of the previous parts distribution (2007, 2008, or 2009) and/or use some DNA that you can easily amplify from readily available tissue by PCR (plant tissues are generally most easy to come by). In addition to the particular part, module, or device your team wants to produce, each team needs to make sure that the new part is made 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. Figuring out how to do this can be quite tricky. Therefore, think about an easy functional assay and discuss it with your teacher. After doing all the work, each team will write a 10+ page summary report (a summary presentation is optional). This report should be done in form of a journal article. It is worth 20% of your grade and has to be submitted during finals week. The report must be uploaded (as MS-Word or PDF) to your project homepage for consideration. No late submissions can be accepted.

How to Start

1) Select a part, module, device, or system to build

Your first team task is to select a project. You may take a look at the iGEM presentations for some ideas and an overview of what undergraduate teams achieved. However, it is completely up to you what you want to do in this class. Please do not get confused by thinking you don't know enough to design a project. Just think about what kind of FUNCTION you want your part to carry out. For example, do you want to produce a certain smell or color? Do you want to build an oscillating device or something that can be activated under certain external triggers? Do you want to build a biological sensor? Or simply convert a plant gene into a biobrick? These are just a few ideas. Do whatever you like to work on. Please do not consider the assumed complexity of your project. We will brake it down to something that is achievable. At this point, JUST FOCUS ON WHAT YOU WOULD LIKE TO WORK ON. Everything else is irrelevant for the design step. Be curious and select something interesting. Don't be afraid to select a project that looks challenging. We will learn something from each project - regardless of how far you get with it. So please select a project that really interests you; something you really would like to do.

Once you found something interesting to work on, find a partner who wants to work together with you or join someone in their project. Browse the parts in the registry to find out what is available to use and what you can build on to achieve your plans. Then, make a list of what needs to be done. We will discuss all proposals in the third week. I will assist you in the planning as much as you need. But you have to come with ideas for discussion and ask specific questions. I will not give you suggestions on what to do, except for the above. And I will not tell you if something is possible or not. Everything is possible and we should not limit ourselves.

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:

  • description of the product (part, module, device, or system)
  • 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 - and the instructor. You have to sit together and play around with 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, how to edit pages, how to link to other information, and how to upload pictures. Just use the "help" function on the left or on top of each page. Do not forget to apply for an account (which is free) as soon as possible. Do it after the first class session. You need the working account very soon. So please don't delay applying. Your experimental design, the approach you take, and the results you expect must be explained in form of a research proposal and uploaded to your project website. This proposal needs approval by the instructor before any 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. A good selection of "need to know" questions is very helpful in developing your experimental approach. Ask yourself: "What information (about the part and the experimental procedures) do I need in order to produce and verify my part/device/system?" Prepare a list of issues you need to address!

Please be aware that the first 2-3 weeks will 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. After you started your bench work, we will have no more lectures or formal instruction. From than on, we devote every minute to practical work.

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 the 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 as much detail as possible (in form of the 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 (due by the end of week 3). However, your proposal should be complete by the end of week two to allow for a review and for making improvements. A complete proposal does not mean that you have to have all answers. You probably won't. I just expect that you have your ideas down and know what you need to do to achieve what you proposed. Each team will present its proposal in a 15 minute presentation in the last session of week 3. This presentation is part of the 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 and 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 MUST 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. It is necessary that everyone writes a good notebook and keeps it updated. The notebook will be worth 30% of your grade. It will be 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. The notebooks can never leave the lab and have to be available any time during class sessions.

In addition to and based 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. Team members should sit together before the end of class to SUMMARIZE their achievements, acknowledging every ones contribution. This includes pictures of important results (for example gel pictures). And 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 team does not accomplish it, the achievements as well as problems must be explained thoroughly in the lab notebooks, online journal, and the final presentaion. 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 build 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 35% 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 15% 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 do the work! Cell phones and other electronic devices (iPhones or MP3 players) are not allowed in the lab and will be confiscated if used.

Final Grade Composition:

  • 10% project proposal and presentation (due by end of week 3)
  • 40% lab notebook
  • 30% individual contribution (ideas presented, preparation for class, performance, knowledge)
  • 20% project summary report (10+ pages)

optional: 10% final summary presentation (15 minutes)

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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