Team:Freiburg/Results

From 2011.igem.org

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=results=
=results=
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
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[http://partsregistry.org/Part:BBa_K608404 BBa_K608404]
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IPTG-inducible Promoter with plastic binding domain-tagged GFP
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[http://partsregistry.org/Part:BBa_K608406 BBa_K608406]
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'''Precipitator'''
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Protein domain of Precipitator. Artificial Leucine Rich Repeat(LRR) with C and N-terminal hagfish domain fragments capping the artifical middle part. This part is one version of three different designed to bind nickel by histidines,
 +
grouped together pointing away from the horseshoe shaped protein.
 +
 +
Bacterial LRR Consensus of the central LRR fragment:
 +
 +
LxxLxLxxNxLxxLPxxLPxx
 +
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Protein code:
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CPSRCSCSGTEIRCNSKGLTSVPTGIPSS
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ATRLELESNKLQSLPHGVFDK
 +
 +
LTQLTKSNNHLHSLPDNLPAS
 +
 +
LEVLDVSNNHLHSLPDNLPAS
 +
 +
LEVLDVSNNHLHSLPDNLPAS
 +
 +
LEVLDVSNNHLHSLPDNLPAS
 +
 +
LEVLDVSNNHLHSLPDNLPAS
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LEVLDVSNNHLHSLPDNLPAS
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LKELALDTNQLKSVPDGIFDR
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LTSLQKIWLHTNPWDCSCPRIDY
 +
 +
LSRWLNKNSQKEQGSAKCSGSGKPVRSIICP
 +
 +
 +
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This protein can be used to complex Nickel or Cobalt.
 +
The principal mechanism is comparable to Ni-NTA columns, as chelates the ions. Free binding sites of the ions are then exposed, so that a His-tagged protein can attach to them.
 +
The design of the protein is of a particular interest, too. LRR are highly conserved motifs throughout evolution. They appear in all kingdoms of life in almost every thinkable role (Ligases, Receptors, Toxins etc.). Their core is highly conserved and provides a very stable backbone, while the intermediate, non-conserved aminoacids are almost freely interchangeable.
 +
 +
 +
This protein can be used to complex up to 4 Nickel or Cobalt. However the principal design oft he protein is of a particular interest, too. LRR are highly conserved motifs throughout evolution. They appear in all kingdoms of life in almost every thinkable role (Ligases, Receptors, Toxins etc.). Their core is highly conserved and provides a very stable backbone, while the intermediate, non-conserved aminoacids are almost freely interchangeable.
 +
 +
We only submitted one of the three versions, to reduce redundancy in the registry. Please contact us for any questions.
 +
 +
 +
<!-- Add more about the biology of this part here
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===Usage and Biology===
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The Precipitator is a new artificially designed LRR protein. It is meant as protein that binds Nickel ions with Histidines grouped on its surface. The bound Nickel can then precipitate His-tagged proteins. In our Lab in a Cell it should function as an adaptor between the plastic surface of pipettes and the His-tagged protein. Please look at our detailed description of the design layout in our modeling section. The sequence was synthesised and cloned into the iGEm vector. The submitted sequence was fully confirmed by sequencing.
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[http://partsregistry.org/Part:BBa_K608407 BBa_K608407]
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===Design Notes===
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Here we investigated an optimal set of non-conserved aminoacids by analysing large sets of similar proteins and databases. You can use this piece of work as a template to design your own protein and give it any function you like, by simply interchanging aminoacids and fusing other domains on the N or C termini.
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To guarantee proper folding and to shield off the hydrophobic core, a well studied fragment of an LRR protein coming from hagfish was used. This technique was investigated before
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===Source===
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The part is constructed from the following protein sequences
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(PDB: 2z66) N-Terminal hagfish
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(PDB: 2z62) C-Terminal hagfish
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(PDB: 3cvr) bacterial ligase
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'''Precipitator fused with GST tag'''
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The Precipitator was fused with the C-terminus of our GST tag in order to extract and further test the construct for its Nickel binding affinity. We successfully cloned it together using the Gibson Assembly and then subsequently pasted it into the iGEM vector. The submitted sequence was partially confirmed by sequencing.
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[http://partsregistry.org/Part:BBa_K608408 BBa_K608408]
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'''GST-tag'''
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The GST-tag was PCR amplified from a pGEX vector with overhang primers including the iGEM restriction sites and then pasted into the iGEM vector. To verify the functionality of the construct we cloned it before a GFP sequence and expressed it with an IPTG inducible vector. Results see partsregistry page. The submitted sequence was partially confirmed by sequencing.
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===Results===
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The non-purified, sonificated supernatant of a 50ml E.coli culture pellet was resuspended in 7.5ml PBS. This sample delivered 319,4 µg/µl of protein detected with a Bradford assay. A second identical sample was GST-purified with a Glutathion-Sepharose pull down assay and resulted in 1ml of sample with a  protein concentration of 1,6 µg/µl.
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However both concentrations were a little above the straight calibration line values. Therefore the results are not perfectly quantified, but the numbers are still significant enough to provide a good proof of the functionality of the assay.
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Then the GFP fluorescence of 100µl of supernatant of both samples was measured with a plate reader:
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Non-purified: 180630
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GST-purified:  243011
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Thus the ratio of GFP absorbance / 1µg protein is:
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Non-purified: 5642
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GST-purified: 1432257
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If the values of the Bradford assay are assumed as correct, the purified sample has a 254x higher GFP concentration than the non-purified sample. Due to 3 washing steps it can be assumed that there is barely any other protein left except GFP.
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Furthermore it can be estimated that only 17% of the total GFP amount present in the 50ml cell culture, could be captured using this assay.
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===Methods===
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Plate reader:
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The fluorescence intensity and protein concentration were measured with the
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FLUOstar Omega, which is a multi-mode microplate reader.
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Samples were pipetted into the microplate and analyzed via the plate
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reader. In this experiment we focused on the protein concentration and the
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fluorescence intensity of GFP.
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Bradford-assay: A method to determine the total protein concentration.
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To analyze the protein concentration of the samples, Coomassie Brillant Blue
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was pippeted to each sample. With the binding of the dye to the proteins the
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color changes from dark red to blue. The more protein in the solution the
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more Coomassie dye can bind to proteins and the more the color changes into
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blue. The absorption of bound Coomassie dye is 595nm. The absorbance is
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proportional with the amount of bound dye. With a series of Bovine Serum
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Albumin (BSA) measurements the exact protein concentration of the samples
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can be determined. BSA acts like a “marker” because the concentration of BSA
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is known and with a linear calibration line the exact protein concentration can
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be detected.
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===Usage and Biology===
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'''Prepare construct'''
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*Cloning: Clone the GST Biobrick part BBa_K608408 beforte the N-terminus of the protein you wish to purify. You may want to put a Glycine linker inbetween the two parts, for better expression and folding proterties of the fusion protein. DO NOT USE THE RFC10 STANDARD, but another, which allows fusion proteins. We recommend the Gibson Assembly, it worked conviently for us.
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*Vector: You may want to use a vector especially designed for protein expression. The iGEM vectors are all high copy plasmids, which can lead to an overload of protein in your E. coli cells. This can result in toxic effects or inclusion bodies.
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*Transformation: you may want to chose a E. coli strain especially designed for protein expression. It is helpful for a successfull protein purification to use a protease deficient strain such as BL21.
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'''Cell culture and Lysis'''
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*Grow cells overnight after transformation in 2ml medium.
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*Inoculate 250mL LB in a 1l flask
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*Optional: induce with IPTG (500 µL)
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*centrifuging cells in 50ml falcon tubes
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*resuspend pellets in 7,5µl ice cold PBS
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*sonificatie cells (4x 1 min with pause, maximal power, 1 pulse per second)
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*Add 1,5X Protease inhibitor and 25µl of 10mM Lysozyme and let rotate at room temperature for 30min
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*Centrifuge lysate for 10min at 500xg
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*Preparation of glutathione sepharose beads. You may add 1X protease inhibitor to all used PBS.
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*Gently shake the bottle of sepharose to resuspend the matrix.
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*Use a pipet to remove sufficient slurry for use and transfer to an 15 ml falcon tube. (Dispense 1.33 ml of original sepharose slurry per ml of final volume required.)
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*Sendiment the matrix by centrifugation at 500xg for 5 min. Carefully decant the supernatant.
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*Wash the sepharose by adding 10 ml of cold 1xPBS per 1.33 ml of the original slurry of glutathione sepharose dispensed. Invert to mix. (Sepharose must be thoroughly washed with PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures).
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*Sediment the matrix by centrifugation at 500xg for 5 minutes. Decant the supernatant.
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*For each 1.33 ml of the original slurry, add 1 ml of 1xPBS. This produces a 50% slurry. Mix well prior to the subsequent pipetting steps. (Sepharose 4B equilibrated with PBS may be stored at 4°C for up to a month.
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'''Purification of fusion proteins'''
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*Transfer supernatant from step 2)h) on top of slurry from step 4)f)
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*Rotate falcon for 30 min
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*Centrifuge at 500xg for 5min
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*Collect supernatant in a separate 50ml falcon ( in case the protein does not attach well to the beads, this supernatant can be used for a second purification)
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*Add 10ml of cold PBS with protease inhibitor to flask with the beads
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*Rotate falcon for 10 min
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*Repeat steps c) - f)
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*Centrifuge at 500xg for 5min
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*Collect supernatant in a separate 50ml falcon (in case the protein does not attach well to the beads, this supernatant can be used for a second purification)
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*Eluate beads with 1ml elution buffer (50mM Tris pH 8, 10mM Glutathione) – transfer slurry into a 2ml eppi
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*Centrifuge eppi at 13000rpm to make a stable pellet
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*Transfer supernatant to a new eppi. Take care not to take up beads from the pellet.
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*Optional: repeat j) – l) to wash the rest off the beads
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===Further reading===
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Sandra Harper, David W. Speicher  (2008); „Expression and Purification of GST Fusion Proteins“; John Wiley and Sons, Inc.; DOI: 10.1002/0471140864.ps0606s52
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==<span style="color:green;">Green light receptor</span>==
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[http://partsregistry.org/Part:BBa_K608101 BBa_K608101]
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'''CcaR''', green light response regulator
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The green light response regulator CcaR was amplified via PCR from<br/>
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the whole ''Synechocystis sp. PCC 6803'' genome and inserted into the pSB1C3 vector.
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[http://partsregistry.org/Part:BBa_K608102 BBa_K608102]
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CcaS, green light receptor
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[http://partsregistry.org/Part:BBa_K608151 BBa_K608151]
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translational unit of pcyA
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==<span style="color:orange;">Lysis cassette</span>==
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[http://partsregistry.org/Part:BBa_K608351 BBa_K608351]
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(correct) temperature sensitive promoter
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[http://partsregistry.org/Part:BBa_K608352 BBa_K608352]
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Bacteriophage Lysis Cassette with RBS
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==<span style="color:black;">commons</span>==
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At the beginning we had to assemble promoters and ribosome binding sites of different "strengths" (ie consensus identities) in order to ensure proper and controlled expression of our parts.<br/>
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<br/>
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The cloning procedure was time-consuming (circa two weeks), so we decided to<br/>
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send our constructs to the registry. Herewith we hope to help others save some time.<br/>
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We called these constructs ''PR'' to abbreviate "Promoter and RBS"
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[http://partsregistry.org/Part:BBa_K608002 BBa_K608002]
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strong Promoter and strong RBS
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[http://partsregistry.org/Part:BBa_K608003 BBa_K608003]
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strong Promoter , medium RBS
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[http://partsregistry.org/Part:BBa_K608004 BBa_K608004]
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strong Promoter , weak RBS
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[http://partsregistry.org/Part:BBa_K608005 BBa_K608005]
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medium Promoter , strong RBS
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[http://partsregistry.org/Part:BBa_K608006 BBa_K608006]
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medium Promoter , medium RBS
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[http://partsregistry.org/Part:BBa_K608007 BBa_K608007]
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medium Promoter , weak RBS
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----
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To quantify the strength of our PR-constructs we cloned GFP and RFP behind it<br/>
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and quantified the protein output.<br/>
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[http://partsregistry.org/Part:BBa_K608008 BBa_K608008]
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constitutive strong promoter with medium RBS and GFP
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[http://partsregistry.org/Part:BBa_K608009 BBa_K608009]
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Strong promoter with weak RBS and GFP
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[http://partsregistry.org/Part:BBa_K608010 BBa_K608010]
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Medium promoter with strong RBS and GFP
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[http://partsregistry.org/Part:BBa_K608011 BBa_K608011]
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Medium promoter with medium RBS and GFP
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[http://partsregistry.org/Part:BBa_K608012 BBa_K608012]
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Medium promoter with weak RBS and GFP
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----
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[http://partsregistry.org/Part:BBa_K608013 BBa_K608013]
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strong Promoter with strong RBS and RFP
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[http://partsregistry.org/Part:BBa_K608014 BBa_K608014 ]
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PR with RFP
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[http://partsregistry.org/Part:BBa_K608015 BBa_K608015]
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PR with RFP
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[http://partsregistry.org/Part:BBa_K608016 BBa_K608016]
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PR with RFP
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[http://partsregistry.org/Part:BBa_K608017 BBa_K608017]
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PR with RFP
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[http://partsregistry.org/Part:BBa_K608018 BBa_K608018]
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PR with RFP
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{{:Team:Freiburg/Templates/footer}}
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===<span style="color:grey;">Plastic binding domain</span>===
===<span style="color:grey;">Plastic binding domain</span>===

Revision as of 00:32, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

results

Precipitator

BBa_K608404 IPTG-inducible Promoter with plastic binding domain-tagged GFP

BBa_K608406 Precipitator


Protein domain of Precipitator. Artificial Leucine Rich Repeat(LRR) with C and N-terminal hagfish domain fragments capping the artifical middle part. This part is one version of three different designed to bind nickel by histidines, grouped together pointing away from the horseshoe shaped protein.

Bacterial LRR Consensus of the central LRR fragment:

LxxLxLxxNxLxxLPxxLPxx

Protein code:

CPSRCSCSGTEIRCNSKGLTSVPTGIPSS

ATRLELESNKLQSLPHGVFDK

LTQLTKSNNHLHSLPDNLPAS

LEVLDVSNNHLHSLPDNLPAS

LEVLDVSNNHLHSLPDNLPAS

LEVLDVSNNHLHSLPDNLPAS

LEVLDVSNNHLHSLPDNLPAS

LEVLDVSNNHLHSLPDNLPAS

LKELALDTNQLKSVPDGIFDR

LTSLQKIWLHTNPWDCSCPRIDY

LSRWLNKNSQKEQGSAKCSGSGKPVRSIICP


This protein can be used to complex Nickel or Cobalt. The principal mechanism is comparable to Ni-NTA columns, as chelates the ions. Free binding sites of the ions are then exposed, so that a His-tagged protein can attach to them. The design of the protein is of a particular interest, too. LRR are highly conserved motifs throughout evolution. They appear in all kingdoms of life in almost every thinkable role (Ligases, Receptors, Toxins etc.). Their core is highly conserved and provides a very stable backbone, while the intermediate, non-conserved aminoacids are almost freely interchangeable.


This protein can be used to complex up to 4 Nickel or Cobalt. However the principal design oft he protein is of a particular interest, too. LRR are highly conserved motifs throughout evolution. They appear in all kingdoms of life in almost every thinkable role (Ligases, Receptors, Toxins etc.). Their core is highly conserved and provides a very stable backbone, while the intermediate, non-conserved aminoacids are almost freely interchangeable.

We only submitted one of the three versions, to reduce redundancy in the registry. Please contact us for any questions.


Retrieved from "http://2011.igem.org/Team:Freiburg/Results"