Project update week 4

Welcome to my logbook and documentation page ;)
Currently, I am thinking of 3 projects

  • 1 Restriction enzymes
  • 2 Synthetic DNA
  • 3 DIY webcam

Project 1 Restriction enzymes

Yeah! I orderded the Biorad kit: http://www.bio-rad.com/en-nl/product/restriction-digestion-analysis-lambda-dna-kit .

Plan

  • See how the Restriction Digestion and Analysis of Lambda DNA Kit works
  • Do the experiment in the Waag in Amsterdam
  • See if I can do the experiment in my kitchen at home

The lambda phage is a bacterial virus that infects E.coli: https://en.wikipedia.org/wiki/Lambda_phage.

The bacterial virus has approximately 48,000 base pairs.

How does the kit work?

  • Each restriction enzyme used in this kit cuts the lambda DNA several times, generating distinct sets of DNA restriction fragments of different sizes.
  • The three different sets of DNA fragments that result from the enzymatic digestion are separated by agarose gel electrophoresis and visualized.
  • Compare the banding patterns from each sample to each other and to a DNA size standard.
  • Use the electrophorsis results to construct standard curves and determine the precise DNA fragment sizes generated by the different restriction enzymes.

More information about restriction enzymes

This is a very helpful video about restriction enzymes https://www.youtube.com/watch?v=U2cKywEn6KY

Notes

  • Restriction enzymes (RE) are a protection mechanism of bacteria against virusses
  • The RE needs to recognize a certain sequence
  • For example, Ecor1 recognizes a palindromic sequence
  • It cuts in non methylated sequences, because these are not from the bacteria itself
  • The RE cuts DNA open and leaves so-called sticky ends
  • Possible applications are making synthetic insulin by putting the insulin gene into the open part of the bacteria gene

This is a very helpful video about molecular biology https://www.youtube.com/watch?v=yYIZgS-L5Sc

Notes

  • TAQ polymorase discovered in Yellowstone park
  • Quote: Genetic engineering is build upon this method (but is was before CRISPR/cas9)
  • Analogy of ransom note: scissors are RE, glue are hydrogen bonds, liner is gel electroprosis, copier is PCR and human eye is DNA sequencing
  • An example of a RE is Hind3 https://en.wikipedia.org/wiki/HindIII which is also in the Bio-rad kit!
  • Hind3 is different compared to Ecor1, because it cuts at a different place.
  • What I do not understand, what is the role of the primer in TAQ polymorase? Probably to be used in PCR to copy and cut? https://en.wikipedia.org/wiki/Taq_polymerase

Next steps

These are the next steps:

  • Ask Roland if the kit is allowed in the Waag (probably it is, because I am not making an GMO)
  • Fill in the form and order the kit

Project 2 Synthetic DNA

I am fascinated by the possibilities of synthetic biology.

Next steps

These are the next steps:

  • Ask Roland if I can order the synthetic DNA?
  • Answer = yes. Tip: order at Eurofins or Baseclear
  • Decide on which translation method to use?
  • Answer = maybe this one http://montessorimuddle.org/2013/02/02/dna-writer/
  • Discuss what I can do with the synthetic DNA
  • Answer = make multiple copies of the text

Notes

Project 3 DIY microscope

Making labproducts

Next steps

These are the next steps:

  • Download the .stl files
  • Download Cura program
  • Export to j-code
  • Print
  • Buy webcam and other materials
  • Ask fablab experts to help me with the setup

Presentation

For the presentation I am thinking of different things:

  • present the difference between restriction enzymes and CRISPR/cas9
  • see how I can continu the process at home

This will probably be in the form of a presentation, audio and/or video file.

Written on February 18, 2019