Team:Heidelberg/Toolbox

iGEM TEAM HEIDELBERG 2017

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COLLAPSE NAVIGATION

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• People
  • Team
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• Project
  • Overview
  • Background & Design
  • PACE
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  • Results Summary
  • Software Validation
  • Organosilicons
  • Protein Interaction
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• Parts
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• Modeling
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  • Mutagenesis Induction
  • Mutation Rate Estimation
  • Lagoon Contamination
  • Medium Consumption
  • Interactive Web Tools
• Software
  • AiGEM Overview
  • DeeProtein
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• Toolbox Guide

Welcome to our
Toolbox Guide!

To enable rapid design and simple cloning of APs, we created a software tool, so that cloning can easily be planned. All parts that can be chosen are available in pSB1C3 backbones from the registry. Just follow the instructions and create your own fully customized AP.

Ready to PREDCEL?

See our Questionnaire here

Have you already chosen a protein to be evolved?

Yes

No

You already accomplished the first and most important step. Let’s see whether you are ready to evolve it!

No problem, there are lots of proteins waiting to be evolved.Go on to find out what you should take care of choosing your protein and preparing your experiments. Maybe have a look at our toolbox to get an idea of the wide range of proteins you can evolve with PREDCEL

Which approach do you pursue by using our evolutionary toolbox?

Medical application

Chemical synthesis

Agricultural approach

Could be helpful somehow

Other scientific interests

Did you discuss your idea with a medical professional? We are sure this expertise would be valuable to choose the right experimental settings.

Did you consider that your products might be dangerous? To get to know this, you could engage a chemical expert.

Be aware that a number of countries have strict regulations regarding green biotechnology.

That sounds quite uncertain. Before you start or pursue evolving your protein, you should know the exact purpose and impact. How about engaging with your local community to figure out possible needs?

Engage with the community in the field of in vivo evolution – we´d be interested in discussing your project.

Are you sure your protein is safe to be evolved?

Yes it is safe!

How can I know?

Nevertheless we advise you to check it in our SafetyNet, there could be “sleeping” hazardous potential.

Check our SafetyNet

Do you know which equipment is required to safely work with phages?

Yes

Not sure

No

Did your considerations include that phages are very volatile? You should create a special “phage space” in your lab to reduce contamination. Furthermore, you should comply with the safety level of your evolving protein. Check the safety level of your protein on our SafetyNet

Check advices on our safety page and get in touch with your safety representative

Before performing your PREDCEL experiments you should reflect on the consequences

Did you calculate all required parameters on our Integrated Modeling page?

Yes

Not yet

I don´t intend to

Well done! Now you have the optimal conditions to succeed

Visit our Integrated Modeling page to figure out all required parameters for a successful PREDCEL run

You should consider that our Integrated Modeling reduces effort, costs and environmental burden

Did you already engage with your local community to discuss your plans?

Yes

Not yet

I don´t intend to

Fine. For more ideas, how to engage with the public you could watch the expert interview with Dr. Kaufmann

You should integrate public needs to ensure the beneficial impact for humanity of your research aim

You should first engage with your local community to discuss concerns before using our PREDCEL toolbox

Did you consider possible consequences of your planned PREDCEL experiment?

Yes

Not yet

I don´t intend to

You are a responsible scientist. We encourage you to use our PREDCEL toolbox and convey the spirit of responsible science to your friends and colleagues

Take your time to reflect and research on possible consequences to prevent endangering the environment and yourself. You should engage with experts in this field

Before starting your experiment, you should consult experts and their recommendations

Now it´s on you!

When you talk about PREDCEL with your colleagues, friends and local communities, you can spread the idea of making directed evolution experiments safe and to use it responsibly for addressing urgent human needs

1. Optimize parental sequence / gene pool

2. Get phage

3. Add selection pressure

1. First, choose your preferred backbone from the pull down menu. We provide three different origins of replication that differ in their copy number and therefore affect the selection stringency, your AP will have. Be aware that the origin of replication should be compatible to the origin of replication of all other plasmids you plan to use. In most of the cases it is best to combine your origin of replication with an ampicillin resistance, but we provide other antibiotic resistances for special applications as well.

2. Second, decide which RBS you need for control of geneIII translation. We offer six different RBS with different strengths. For further information look...

3. Next, decide, which reporter you would like to choose. A set of different fluorescent and luminescent reporters is available

Plasmid

Resistance & Ori

Select

RBS + geneIII

Select

RBS + Reporter

Select

Activator & Promoter

4. When all standard parts are defined, the promoter and associated regulatory sequences must be set. Decide between the given options. Enter either an own sequence (as text or upload the file) or use one of the provided standard promoters that were already used by our team. Notice that the length of this part should not be below 200 bp. Ensure, that your sequence does not include a RBS.

Select

Overlaps are included

Overlaps are included

Additional sequence

5. Finally, decide which additional gene you would like to express on your AP. Most circuits that are used in PACE, need at least one additional protein, like split-proteins, chaperone or interacting factors. Again, it is possible to enter the desired sequence or choose one of the provided genes. Make sure, you enter a whole expression cassette, with promter, RBS, CDS and terminator.

Select

Overlaps are included

Overlaps are included

Create Plasmid

Instructions

4. Induce mutagenesis

5. Optimize conditions