In Freiburg
2011, in the nice and warm springtime, I hosted the first DIY Biohacking
session in Germany. We were four people with backgrounds in biology,
electronic engineering, informatics and journalism.
P. crafted a powersupply for Gelelectrophorese and we did several successfull
testruns in comparison with a professional machine. Sascha Karberg tried several DNA extraction kits on oral mucosa cells from P.
and me. Afterwards we ran a PCR with Primers typically used in forensic laboratories
for genetic fingerprinting and resolved the output on Agarose-gels, thereby experimenting with the DNA
stains Cyber-safe and Methylene Blue.Cyber-safe was far more effective, but requires UV exitation, while methylene
blue is visible in daylight (but barely only, not recommendable). The PCR
failed due to wrong primer conenctrations. On the side I tried to decipher the menu setup of my Old-school COY
thermocycler from the 80ies and collected temperature curves of the cycles,
with the help of a little arduino based thermometer.
Special thanks to Dr. Friedel Drepper, Biochemistry Department, Faculty for
Biology II, University of Freiburg, for his generous support and advice for my home lab!
We aim to transform protein expression in bacterial systems into an
elegant, fast and affordable process.
By eliminating routine use of expensive materials, this novel assay will
utilize sustainable laboratory equipment and widespread His-Tag
technology.
We propose an expression system induced by blue, green and red
light, combined with subsequent temperature controlled autolysis of E. coli.
The purification of the his-tagged protein of interest will be
accomplished by
an adaptor protein of our own design which binds the His-Tag on one side
and the surface of serological pipettes on the other. Two subsequent
pipetting steps for washing and purification of the cell lysate will
quickly elute the product.
Our system will provide expression and purification of Polymerase
and Ligase, but will be easily expandable to any His-tagged protein
needed by the modern molecular biologist.
Gene technology is driven by the use of restriction
endonucleases.
Yet, constraints of limited sequence length and variation recognized by
available restriction enzymes pose a major roadblock for synthetic
biology. We developed the basis for universal restriction enzymes,
primarily for routine cloning but also with potential for in vivo
applications. We use a nucleotide cleavage domain fused to a binding
domain, which recognizes a programmable adapter that mediates binding
to DNA and thus cleavage. As adapter we use readily available modified
oligonucleotides, as binding domain anticalins and as cleavage domain
FokI moieties engineered for heterodimerization and activity. For
cloning, this universal enzyme has merely to be mixed with the sequence
specific oligonucleotide and the target DNA. Binding and release are
addressed with thermocycling. Additionally, we provide concepts for in
vivo applications by external adapter delivery, activity regulation by
photo-switching, as well as for modifying an argonaute protein towards
a DNA endonuclease.
Gene technology is driven by the use of restriction
endonucleases.
Yet, constraints of limited sequence length and variation recognized by
available restriction enzymes pose a major roadblock for synthetic
biology. We developed the basis for universal restriction enzymes,
primarily for routine cloning but also with potential for in vivo
applications. We use a nucleotide cleavage domain fused to a binding
domain, which recognizes a programmable adapter that mediates binding
to DNA and thus cleavage. As adapter we use readily available modified
oligonucleotides, as binding domain anticalins and as cleavage domain
FokI moieties engineered for heterodimerization and activity. For
cloning, this universal enzyme has merely to be mixed with the sequence
specific oligonucleotide and the target DNA. Binding and release are
addressed with thermocycling. Additionally, we provide concepts for in
vivo applications by external adapter delivery, activity regulation by
photo-switching, as well as for modifying an argonaute protein towards
a DNA endonuclease.
