Bacon ipsum dolor amet kielbasa ea boudin pig picanha anim. Jowl ullamco short ribs turducken enim in. Cillum shankle ut in t-bone meatball aliqua sed. Tempor ad non nostrud veniam chicken biltong meatball aliqua tri-tip lorem exercitation. Excepteur quis sausage irure incididunt andouille deserunt duis alcatra. Veniam fatback non, pork nisi dolor pancetta ullamco shank sed officia exercitation commodo. In tail eu exercitation, dolor pariatur capicola consequat.
Aliqua sausage turducken, reprehenderit culpa chicken capicola frankfurter spare ribs proident brisket porchetta. Bresaola in kevin corned beef, dolor aliquip consectetur. Porchetta pastrami beef ribs ex spare ribs pariatur corned beef alcatra kevin filet mignon laboris dolore jerky nulla. Cillum short ribs tongue laboris. Nulla adipisicing ut esse non ut veniam. Consectetur cupim jerky ullamco. Jerky brisket incididunt in.
Actual introduction
Aliqua xxdh001xx sausage turducken, reprehenderit culpa chicken capicola frankfurter spare ribs proident brisket porchetta. Bresaola in kevin corned beef, dolor aliquip consectetur. Porchetta pastrami beef ribs ex spare ribs pariatur corned beef alcatra kevin filet mignon laboris dolore jerky nulla. Cillum short ribs tongue laboris. Nulla adipisicing ut esse non ut veniam. Consectetur cupim jerky ullamco. Jerky brisket incididunt in.
Turkey turducken dolor tempor tail, in sunt do kevin beef ribeye. Laborum frankfurter pig, consequat leberkas chicken drumstick ut short ribs. Pancetta magna voluptate sausage pork chop ut ipsum officia consectetur bresaola mollit beef ham hock quis deserunt. Sed magna sunt venison capicola boudin cow anim t-bone salami cupidatat kevin shoulder chuck.
Landjaeger mollit swine, in meatball ut cupidatat sed jowl dolore tail laboris voluptate beef sirloin. Pork chop magna shoulder tempor pork. Pork chop tenderloin veniam venison pariatur nulla, ut quis sunt salami cupidatat adipisicing irure ribeye short ribs. Et do excepteur quis proident ipsum pork chop aliquip mollit prosciutto cow cillum. Turkey alcatra leberkas chicken ipsum dolor. Fugiat ea beef filet mignon tongue cow cupim ball tip voluptate eiusmod pork chop. Jerky leberkas boudin nostrud, ut ullamco aliqua frankfurter cupidatat pariatur labore.
Aliqua sausage turducken, reprehenderit culpa chicken capicola frankfurter spare ribs proident brisket porchetta. Bresaola in kevin corned beef, dolor aliquip consectetur. Porchetta pastrami beef ribs ex spare ribs pariatur corned beef alcatra kevin filet mignon laboris dolore jerky nulla. Cillum short ribs tongue laboris. Nulla adipisicing ut esse non ut veniam. Consectetur cupim jerky ullamco. Jerky brisket incididunt in.
Turkey turducken dolor tempor tail, in sunt do kevin beef ribeye. Laborum frankfurter pig, consequat leberkas chicken drumstick ut short ribs. Pancetta magna voluptate sausage pork chop ut ipsum officia consectetur bresaola mollit beef ham hock quis deserunt. Sed magna sunt venison capicola boudin cow anim t-bone salami cupidatat kevin shoulder chuck.
Landjaeger mollit swine, in meatball ut cupidatat sed jowl dolore tail laboris voluptate beef sirloin. Pork chop magna shoulder tempor pork. Pork chop tenderloin veniam venison pariatur nulla, ut quis sunt salami cupidatat adipisicing irure ribeye short ribs. Et do excepteur quis proident ipsum pork chop aliquip mollit prosciutto cow cillum. Turkey alcatra leberkas chicken ipsum dolor. Fugiat ea beef filet mignon tongue cow cupim ball tip voluptate eiusmod pork chop. Jerky leberkas boudin nostrud, ut ullamco aliqua frankfurter cupidatat pariatur labore.
Aliqua sausage turducken, reprehenderit culpa chicken capicola frankfurter spare ribs proident brisket porchetta. Bresaola in kevin corned beef, dolor aliquip consectetur. Porchetta pastrami beef ribs ex spare ribs pariatur corned beef alcatra kevin filet mignon laboris dolore jerky nulla. Cillum short ribs tongue laboris. Nulla adipisicing ut esse non ut veniam. Consectetur cupim jerky ullamco. Jerky brisket incididunt in.
Turkey turducken dolor tempor tail, in sunt do kevin beef ribeye. Laborum frankfurter pig, consequat leberkas chicken drumstick ut short ribs. Pancetta magna voluptate sausage pork chop ut ipsum officia consectetur bresaola mollit beef ham hock quis deserunt. Sed magna sunt venison capicola boudin cow anim t-bone salami cupidatat kevin shoulder chuck.
Landjaeger mollit swine, in meatball ut cupidatat sed jowl dolore tail laboris voluptate beef sirloin. Pork chop magna shoulder tempor pork. Pork chop tenderloin veniam venison pariatur nulla, ut quis sunt salami cupidatat adipisicing irure ribeye short ribs. Et do excepteur quis proident ipsum pork chop aliquip mollit prosciutto cow cillum. Turkey alcatra leberkas chicken ipsum dolor. Fugiat ea beef filet mignon tongue cow cupim ball tip voluptate eiusmod pork chop. Jerky leberkas boudin nostrud, ut ullamco aliqua frankfurter cupidatat pariatur labore. Aptabody
Important graph
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Short introduction
“The sea is everything. […] The sea is the vast reservoir of Nature. The globe began with sea, so to
speak”, Nemo said while the “Nautilus” was cruising with a school of hammerhead sharks deep
beneath the waves.
And the captain was right. Deep in the ocean billions of years ago the miracle of nature took place as
a pool of small molecules evolved to self-replicating lifeforms. The flagship role in this development
was probably taken by the most versatile class of molecules in the history of life: RNA.
Seemingly random nucleotides happened to be in the right order to form the first biocatalysts that
made life on the blue planet possible. Today we know those miracles of nature as ribozymes. Inspired
by this, humanity took evolution into their own hands to create aptamers – nucleic acids capable of
encaging molecules. This allows for the detection of virtually anything. Still this process has been
tedious and time consuming much like fishing with a rod in an ocean. We want to revolutionize this
former evolutionary process and want to make it swift like a shark tracking down its prey.
Yet to really bring out the strengths of these simple yet powerful molecules just comprised of A, U, C
and G we want to combine aptamers and ribozymes to create a toolset for the synthetic biologist to
create allosteric ribozymes able to sense a variety of molecules. Therefore, we hope to introduce the
true origins of life and the capabilities of functional RNA to iGEM.
Join us as we sail forth into new
waters of synthetic biology.
Further content
Test
Test
Notebook
Collecting impressions from the community
It’s done. We’ve finally decided upon a logo. It’s beautiful, it’s blue, it’s fishy *wink*. The next step was to choose a color scheme and a general style for the wiki. As we feared that our own ideas might be too similar we thought of asking a broader public how they would design a website based on our logo. In order to do so, we headed to the ’Neckarwiese’. During summer, a big part of Heidelbergs population decideds to go there and enjoy the sun, have a BBQ or just relax – so we caught them off guard.
We have had prepared a little presentatio with which we introduced them to iGEM and Synthetic Biology. Afterwards we explained them what we planned on doing, showed them our logo and asked, how they would design a website, based on what they’ve just heard. The general consens was to go for a blue theme, maybe add a little algae green and generally stick to maritime iconography. Unexpectedly we actually met someone who knew his stuff: Natalie, a student of the history of arts from Berlin. She gave us useful advice on how play with the different colors, what to avoid and even added a little touch of history to it. Lucky catch!
With fresh ideas we headed back to the lab and continued our experiments, being envious of all the people that could continue to sleep in the sun.
We’ve been lucky and actually met someone knowledgable in arts. Her name is Nati and she studies the history of art in Berlin.
Methods
Extension PCR
Extension PCR was performed using Phusion Flash master mix with equimolar amounts of overlapping oligo DNA and 5 % DMSO.
In vitro Transcription
dsDNA was transcribed into sRNA with T7 RNA polymerase in transcription buffer in the presence of 4 mM ATP, GTP, CTP, UTP each, 10 mM DTT and 5 % DMSO. Reaction was incubated at 37 °C for ~3 h. Then DNase I was added an the reaction was further incubated at 37 °C for 20 min. RNA of interested was purified in 10 % denaturing PAGE. Band were visualized by UV shadowing and appropriate band was excised and eluted using 0.3 M NaAc pH 5.5.
3’ Modifiction of sRNA
Various amounts of sRNA (1-20 µM) were incubated with modified NTPs (150-400 µM) (Biotin-, Alkyne-, Azide-NTP) using either yeast Poly A Polymerase (PAP), affimetrix or Terminale dinucleotidyl Transferase (TdT), NEB in supplied buffers. Reaction was precipitated. Success was check via CuAAC.
Precipitation of nucleic acids
RNA/DNA was precipitated in the presence of 0.3 M NaAc pH 5.5 at -20 °C by the addition of 2.5 volumes of -20 °C cold EtOH. After storing sample at –20 °C for at least 1.5 h is was centrifuged at 14,000 g. Supernatant was removed and pellet was dissolved in H2O. The concentration of the sample was determined using a NanoDrop and calculated using the extinction coefficient from idt oligoanalyzer.
Copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC)
Copper click reaction was performed as described in Winz, 2012. Success was check on denaturing PAGE that was first scanned in appropriate fluorescent mode and after staining with SYBR Gold in SYBR Gold mode on a Typhoon Scanner, GE.
Initial DNAzyme Activity
DNAzyme activity was initially tested by incubating 0.5- 5 µM DNAzyme with 200nM Substrate in DNAzyme buffer at 37 °C for 1.5 h. Then 2 µL (to 25 µL reaction) were added and DNAzyme was digested for 20 min at 37 °C. Reaction was separated on 20 % denaturing PAGE and visualized with SYBR Gold.
Denaturing Polyacrylamid gel electrophorese (PAGE)
Polyacrylamide gels were prepared using Rotiphorese Sequencing Gel System according to manual. Gel were run in TBE buffer. RNA or DNA was visualized by UV shadowing or SYBR Gold stain and scanning with a Typhoon Scanner, GE.
PCR
PCR was performed using Phusion Flash Master Mix (Thermo Scientific), OneTaq® Quick-Load® 2X Master Mix (NEB), Q5® High-Fidelity 2X Master Mix (NEB) or Velocity DNA Polymerase (Bioline) according to manufacturer’s protocol.
PCR Purification
PCR was purified using QIAquick PCR Purification kit from Qiagen according to manufacturer’s protocol.
Gel Extraction
To extract DNA from agarose gel sample was run on a 0.8 % gel in TAE buffer. DNA was visualized under UV using EtBr. Suitable bands were excised and DNA was extracted using QIAquick Gel Extraction kit according to manufacturer’s protocol.
Agarose gel electrophorese
To check the size of DNA fragments or to purify DNA samples were run on a 0.8-4 % agarose gel. To prepare the gel 0.8-4 % (w/v) agarose were heated in TAE or TBE buffer in the microwave. Gel was poured containing ethidium bromide or Roti Gel Stain and visualized by UV light.
Digest of DNA with restriction enzymes
DNA was incubated with restriction enzyme in appropriate buffer and incubated as described in the manual. If necessary enzymes were heat inactivated and afterwards purified by precipitation or PCR purification kit.
Initial DNAzyme Activity
DNAzyme activity was initially tested by incubating 0.5- 5 µM DNAzyme with 200nM Substrate in DNAzyme buffer at 37 °C for 1.5 h. Then 2 µL (to 25 µL reaction) were added and DNAzyme was digested for 20 min at 37 °C. Reaction was separated on 20 % denaturing PAGE and visualized with SYBR Gold.
Ligation
DNA with matching sticky ends were ligated using T4 DNA Ligase in supplied buffer.
Cryostock of E. coli strain
500 µL of oN culture was mixed with 500 µL of autoclaved 40 % glycerol and stored at -80 °C.
Plasmid prep
Plasmids were purified from liquid cultures using QIAprep Mini-, Midi or Maxiprep according to manufacturer’s protocol. Concentration was determined with a NanoDrop.
Python
Python is a general purpose, interpreted, cross-platform, object-oriented, open-source programming language widely used in the scientific world.
OpenMM
We used OpenMM for molecular simulation, specifically to compute the energy levels of given systems consisting of a ligand and an aptamere.
NumPy
NumPy is a fundamental package for scientific computing with Python, version 1.9.2 has been used to hel us doing math.
Amber
Amber is a toolkit for molecular diynamic computations.
pexpect
pexpect is a Python module for handling child applications.
mpmath
mpmath is a Python library for real and complex floating point arithmetic with arbitrary precicion.
ms007forward T7 promotor with Spinach2 containing the c-di-GMP AptamerGGGCTAATACGACTCACTATAGGATGTAACTGAATGAAATGGTGAAGGACGGGTCCACACGCACAGGGCAAACCATTCGAAAGAGTGGGACGCAAAGCCTCCGGCCTAAACCAGAAGACATGGTAGGTAGCGGGGTTACCGATGTTGTTGAGTAGAGTGTGAGCTCCGTAACTAGTTACATC
ms008GATGTAACTAGTTACGGAGC
ms009GGGCTAATACGACTCACTATAGG
ms010reverse Spinach2 containing the c-di-GMP Aptamer MutantGGGCTAATACGACTCACTATAGGATGTAACTGAATGAAATGGTGAAGGACGGGTCCACACGCACAGGGCAAACCGTTCGAAAGAGTGGGACGCAAAGCCTCCGGCCTAAACCAGAAGACATGGTAGGTAGCGGGGTTACCGATGTTGTTGAGTAGAGTGTGAGCTCCGTAACTAGTTACATC
ms011(13)_reverse ATP AptamerGATGTAACTAGTTACGGAGCTCACACTCTACTCAACAAACACATGGACTCCTTCCATGTAGCTCCCGTAAGAGCCCTGAGATCCGTAGATCTCCCTGGACCCGTCCTTCACCATTTC
ms011reverse Spinach2 containing the ATP AptamerGATGTAACTAGTTACGGAGCTCACACTCTACTCAACAAACACATGGACTCCTTCCATGTAGCTCCCGTAAGAGCCCTGAGATCCGTAGATCTCCCTGGACCCGTCCTTCACCATTTC
ms012reverse ATP Spinach with computer generated stem 1GATGTAACTAGTTACGGAGCTCACACTCTACTCAACAAGGGCAAAGACACATGGACTCCTTCCATGTAGCTCCCGTAAGAGCCCTGAGATCCGTAGATCTCCCCCTGGCCCTGGACCCGTCCTTCACCATTTC
ms013reverse ATP Spinach with computer generated stem 2GATGTAACTAGTTACGGAGCTCACACTCTACTCAACAAGGTACAATACACATGGACTCCTTCCATGTAGCTCCCGTAAGAGCCCTGAGATCCGTAGATCTCCCTACGCACCTGGACCCGTCCTTCACCATTTC
ms014forward T7 promotor with Malachite Green AptamerTAATACGACTCACTATAGGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC
ms015reverse T7 promotor with Malachite Green AptamerGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCTATAGTGAGTCGTATTA
ms016forward EcoRV site, T7 promotor and Spinach2 with ATP AptamerGATATCGCGCGCTAATACGACTCACTATAGGATGTAACTGAATGAAATGGTGAAGGACGGGTCCA
ms017reverse HDV and Spinach2 with ATP AptamerGGAGGCTGGGACCATGCCGGCCGATGTAACTAGTTACGGAGCTCACACTCTACTCAACAAACACATGGACTCCTTCCATGTAGCTCCCGTAAGAGCCCTGAGATCCGTAGATCTCCCTGGACCCGTCCTTCACCATTTC
ms023forward T7 2.5 promotor with Malachite Green AptamerTAATACGACTCACTATTAGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC
ms024reverse T7 2.5 promotor with Malachite Green AptamerGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCTAATAGTGAGTCGTATTA
ms025forward Sp6 promotor with Malachite Green AptamerTCATTTAGGTGACACTATAGGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC
ms026reverse Sp6 promotor with Malchite Green AptamerGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCTATAGTGTCACCTAAATGA
ms027forward T3 promotor with Malachite Green AptamerCAATTAACCCTCACTAAAGGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC
ms028reverse T3 promotor with Malachite Green AptamerGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCTTTAGTGAGGGTTAATTG
ms029forward E. coli (T7A1) promotor with Malachite GreenGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCCCTCTCGATGGCTGTAAGTATCCTATAGGTTAGACTTTAAGTCAATACTCTTTTTGATAA
ms030reverse E. coli (T7A1) promotor with Malachite GreenTTATCAAAAAGAGTATTGACTTAAAGTCTAACCTATAGGATACTTACAGCCATCGAGAGGGGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC
ms031forward EcoRV site, T7 promotor and Hammerhead RibozymeGATATCGCGCGCTAATACGACTCACTATAGTCCAGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACTGGT
ms032reverse HDV, Malachite Green Aptamer and Hammerhead RibozymeGGAGGCTGGGACCATGCCGGCCGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCACCAGTTTCGTCCTCACGGACTCATC
ms032reverse HDV, Malachite Green Aptamer and Hammerhead RibozymeGGAGGCTGGGACCATGCCGGCCGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCACCAGTTTCGTCCTCACGGACTCATC
ms034reverse Malachite Green Aptamer with Hammerhead RibozymeGGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCACCAGTTTCGTCCTCACGGA
ms036forward T7 promotor, ATP Aptamer and Hammerhead RibozymeTAATACGACTCACTATAGGAGATCTACGGATCTCAGGGCTCTCCAGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACTGGT
ms037forward EcoRV site, T7 promotor and Spinach2 with ATP Aptamer with computer generated stem 1GATATCGCGCGCTAATACGACTCACTATAGGATGTAACTGAATGAAATGGTGAAGGACGGGTCCAGGGCCAGGGGGAGATCTACGGATCTCAGGGCTCTTACGGGAGC
ms038forward EcoRV site, T7 promotor and Spinach2 with ATP Aptermer with computer generated stem 2GATATCGCGCGCTAATACGACTCACTATAGGATGTAACTGAATGAAATGGTGAAGGACGGGTCCAGGTGCGTAGGGAGATCTACGGATCTCAGGGCTCTTACGGG
ms039reverse HDV and Spinach2 with ATP Aptamer with computer generated stem 1GGAGGCTGGGACCATGCCGGCCGATGTAACTAGTTACGGAGCTCACACTCTACTCAACAAGGGCAAAGACACATGGACTCCTTCCATGTAGCTCCCGTAAGAGCCCTGAG
ms040reverse HDV and Spinach2 with ATP Aptamer with computer generated stemGGAGGCTGGGACCATGCCGGCCGATGTAACTAGTTACGGAGCTCACACTCTACTCAACAAGGTACAATACACATGGACTCCTTCCATGTAGCTCCCGTAAGAGCCCTGAG
ms041reverse Spinach Mutation for RFCAAGCAGCCTACTGGACCCGTCCTTCACCATTTCATTCAGATACATCCTAT
ms042forward Spinach Mutation for RFCCGGCAGCCTACTTGTTGAGTAGAGTGTGAGCTCCGTAACTAGATACATC
