Team:ETHZ Basel/Achievements/BioBrick Toolbox

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= BioBrick Toolbox =
= BioBrick Toolbox =
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To construct the aimed fusion proteins, we first cloned our BioBricks into the storage vector pSEVA132 (Victor de Lorenzo's lab, KanR, pBBR1 ori). All parts are sequence verified and compatible with BBF RFC28 (http://dspace.mit.edu/handle/1721.1/46721).
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[[Image:Part_fusion.png|thumb|500px|'''BioBrick assembly''' A-parts are used for N-Terminal and C-parts for C-terminal fusion. B-part is the linker that connects the two entities.]]
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C-parts enable C-Terminal fusion and N-parts N-Terminal fusion.
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== Chemotaxis BrixBox ==
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To construct the aimed fusion proteins, we first blunt-end cloned our BioBricks into the storage vector pSEVA231 <partinfo>BBa_K422002</partinfo> (Victor de Lorenzo's lab, KanR, pBBR1 ori). This allowed easy sequencing ans storage.
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||BBa_K422003 || CheR || A-part ||
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All parts are compatible with [http://dspace.mit.edu/handle/1721.1/46721 BBF RFC28]. The cloning strategy BBF RFC28 is a method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI. The big advantage of this strategy is that we can simultaneously clone up to 3 different inserts into one single expression vector. Part A can be fused to the Linker Part B which is connected to Part C, the C-terminal part of the fusion protein.
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== Anchor BrixBox ==
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{| class="wikitable sortable" border="0" style="text-align: left"
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|-bgcolor=#cccccc
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|+ align="top, left"|'''Table 1''': Our BioBricks parts for the Chemotaxis, the Anchor, the Light Sensing and the Reporter BrickBox.
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|+ align="bottom"|<nowiki>* compatible for insertion of microRNA binding sites</nowiki>
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|width=100px|ID||width=80px|Insert||width=120px|Orientation||width=100px|ID||width=85px|Insert||width=120px|Orientation||width=500px|Description
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|-
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|<partinfo>BBa_K422003</partinfo>||CheB||A-part||<partinfo>BBa_K422003</partinfo>||CheB||C-part||Demthylase: Demethylates methyl accepting chemotaxis proteins MCPs
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|-
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|<partinfo>BBa_K422005</partinfo>||CheR||A-part||<partinfo>BBa_K422003</partinfo>||CheR||C-part||Methyltransferase: Methylates methyl accepting chemotaxis proteins MCPs
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|-
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|<partinfo>BBa_K422007</partinfo>||CheY||A-part||<partinfo>BBa_K422003</partinfo>||CheY||C-part||Induces tumbling by interacting with the flagellar switch protein FliM
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|-
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|<partinfo>BBa_K422009</partinfo>||tetR||A-part|| || || ||Tetracyclin repressor: Binds to its operator tetO for spatial localization of a fusion protein to the DNA
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|-
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|<partinfo>BBa_K422010</partinfo>||trig||A-part|| || || ||Ribosome binding domain of the trigger factor: Binds to the large subunit of the ribosome
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|-
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| || || ||<partinfo>BBa_K422011</partinfo>||MreB||C-part||Prokaryotic actin homologue: Assembles into helical filaments underneath the cytoplasmic membrane
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|-
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| || || ||<partinfo>BBa_K422012</partinfo>||Pif3||C-part||Phytochrome interacting factor: Binds Prf form of PhyB and rapidly dissociates in response to reconversion to the Pr state
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|-
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| || || ||<partinfo>BBa_K422013</partinfo>||PhyB||C-part||Phytochromes type II: Biologically inactive form Pr absorbs red light and active configuration Pfr far-red light
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|-
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|<partinfo>BBa_K422014</partinfo>||mcyPet||A-part|| || || ||Optimized cyan fluorescent protein CFP, mutation in A206K preventing it from forming dimers
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|-
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|}
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== Ligth sensor BrixBox ==
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= Fusion protein assembly =
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To influence fusion protein ratio in a bacterial cell, two expression vectors (called working vectors) with different origins of replications were generated. Both the repressor protein AraC and the corresponding promoter ParaBAD were inserted into these vectors followed by an insert flanked by AarI-recognition sites. Digest with AarI releases this insert and generates a vector with assembly-compatible overhangs.
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We also gave a lot of thoughts how to make BBF RFC28 (method for combinatorial multi-part assembly based on the Type II restriction enzyme AarI) compatible with Tom Knight's OAS standard. Check out our general scheme for the easy construction of Tom Knight's OAS and BBF RFC28 compatible working vectors [https://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning]!
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{| class="wikitable sortable" border="0" style="text-align: left"
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|-bgcolor=#cccccc
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|+ align="top, left"|'''Table 2''': Our working vectors for fusion protein expression.
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|+ align="bottom"|<nowiki>* compatible for insertion of microRNA binding sites</nowiki>
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|width=100px|ID||width=40px|Name||width=40px|Ori||width=100px|Resistance||width=100px|Source
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|-
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|<partinfo>BBa_K422015</partinfo>||pSEVA132||pBBR1||Amp||Victor de Lorenzo's lab
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|-
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|                                ||pSEVA421||RK2  ||Spec||Victor de Lorenzo's lab
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|-
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|}
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= Characterization of our BioBricks =
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*'''Archeal light receptor fused to bacterial chemotaxis transducer''' <partinfo>BBa_K422001</partinfo>: Details to the construct and protein characterization data can be found under [[Team:ETHZ_Basel/Biology/Implementation|Implementation]]. If you want to see the actual E. lemming epxression the archeal light receptor and therefore reacting to blue light, check out [[Team:ETHZ_Basel/Achievements/The_Lemming|Achievements]].<p style="font-size:2px"><br><br></p>
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*'''Working vector 1 pSEVA132''' <partinfo>BBa_K422015</partinfo>: As pBBR1 is not a widely known origin of replication, the plasmid copy number was determined in relation to a commonly used high-copy vector pUC19. The experimental procedure included normalization of cell number via optical density measurement followed by plasmid concentration measurements (using a commercial Miniprep kit). Details can be found under [[Team:ETHZ_Basel/Biology/Implementation|Implementation]].<p style="font-size:2px"><br><br></p>
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*'''mCyPet''' <partinfo>BBa_K422014</partinfo> has a distinct fluorescence spectra. Measured at an excitation wavelength of 425 nm with a Fluorescein High Precision Monochromator, maximum emission is detected at 482 nm. More information and figures are availabe under  [[Team:ETHZ_Basel/Biology/Implementation|Implementation]].
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= Favourite parts =
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The '''Archaeal light receptor''' <partinfo>BBa_K422001</partinfo> comprising of a fusion of the ''Natronobacterium pharaonis'' Np seven-transmembrane retinylidene photoreceptor sensory rhodopsins II NpSRII and their cognate transducer HtrII to the cytoplasmic domain of the chemotaxis transducer EcTsr of ''Escherichia coli'' is definitely our favourite part. The gene has been codon optimized for expression in E. lemming and subcloned into an IPTG inducible vector. Check out [[Team:ETHZ_Basel/Achievements/E_lemming|our movies]] showing the implementation of this chimeric receptor.
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The other favourite parts are the key element of the E. lemming: The light induced dimerizing domains '''phytochrome interacting factor Pif3''' <partinfo>BBa_K422012</partinfo> and the '''photoreceptor PhyB''' <partinfo>BBa_K422013</partinfo>! Although not present as a fusion construct with a Che-protein and an Anchor-protein yet, these two phytochromes will make the light-switch possible. We are very excited to test this interaction in the short future.

Latest revision as of 18:29, 27 October 2010

BioBrick Toolbox

BioBrick assembly A-parts are used for N-Terminal and C-parts for C-terminal fusion. B-part is the linker that connects the two entities.

To construct the aimed fusion proteins, we first blunt-end cloned our BioBricks into the storage vector pSEVA231 <partinfo>BBa_K422002</partinfo> (Victor de Lorenzo's lab, KanR, pBBR1 ori). This allowed easy sequencing ans storage.

All parts are compatible with [http://dspace.mit.edu/handle/1721.1/46721 BBF RFC28]. The cloning strategy BBF RFC28 is a method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI. The big advantage of this strategy is that we can simultaneously clone up to 3 different inserts into one single expression vector. Part A can be fused to the Linker Part B which is connected to Part C, the C-terminal part of the fusion protein.

Table 1: Our BioBricks parts for the Chemotaxis, the Anchor, the Light Sensing and the Reporter BrickBox. * compatible for insertion of microRNA binding sites
IDInsertOrientationIDInsertOrientationDescription
<partinfo>BBa_K422003</partinfo>CheBA-part<partinfo>BBa_K422003</partinfo>CheBC-partDemthylase: Demethylates methyl accepting chemotaxis proteins MCPs
<partinfo>BBa_K422005</partinfo>CheRA-part<partinfo>BBa_K422003</partinfo>CheRC-partMethyltransferase: Methylates methyl accepting chemotaxis proteins MCPs
<partinfo>BBa_K422007</partinfo>CheYA-part<partinfo>BBa_K422003</partinfo>CheYC-partInduces tumbling by interacting with the flagellar switch protein FliM
<partinfo>BBa_K422009</partinfo>tetRA-part Tetracyclin repressor: Binds to its operator tetO for spatial localization of a fusion protein to the DNA
<partinfo>BBa_K422010</partinfo>trigA-part Ribosome binding domain of the trigger factor: Binds to the large subunit of the ribosome
<partinfo>BBa_K422011</partinfo>MreBC-partProkaryotic actin homologue: Assembles into helical filaments underneath the cytoplasmic membrane
<partinfo>BBa_K422012</partinfo>Pif3C-partPhytochrome interacting factor: Binds Prf form of PhyB and rapidly dissociates in response to reconversion to the Pr state
<partinfo>BBa_K422013</partinfo>PhyBC-partPhytochromes type II: Biologically inactive form Pr absorbs red light and active configuration Pfr far-red light
<partinfo>BBa_K422014</partinfo>mcyPetA-part Optimized cyan fluorescent protein CFP, mutation in A206K preventing it from forming dimers

Fusion protein assembly

To influence fusion protein ratio in a bacterial cell, two expression vectors (called working vectors) with different origins of replications were generated. Both the repressor protein AraC and the corresponding promoter ParaBAD were inserted into these vectors followed by an insert flanked by AarI-recognition sites. Digest with AarI releases this insert and generates a vector with assembly-compatible overhangs.

We also gave a lot of thoughts how to make BBF RFC28 (method for combinatorial multi-part assembly based on the Type II restriction enzyme AarI) compatible with Tom Knight's OAS standard. Check out our general scheme for the easy construction of Tom Knight's OAS and BBF RFC28 compatible working vectors [1]!

Table 2: Our working vectors for fusion protein expression. * compatible for insertion of microRNA binding sites
IDNameOriResistanceSource
<partinfo>BBa_K422015</partinfo>pSEVA132pBBR1AmpVictor de Lorenzo's lab
pSEVA421RK2 SpecVictor de Lorenzo's lab

Characterization of our BioBricks

  • Archeal light receptor fused to bacterial chemotaxis transducer <partinfo>BBa_K422001</partinfo>: Details to the construct and protein characterization data can be found under Implementation. If you want to see the actual E. lemming epxression the archeal light receptor and therefore reacting to blue light, check out Achievements.



  • Working vector 1 pSEVA132 <partinfo>BBa_K422015</partinfo>: As pBBR1 is not a widely known origin of replication, the plasmid copy number was determined in relation to a commonly used high-copy vector pUC19. The experimental procedure included normalization of cell number via optical density measurement followed by plasmid concentration measurements (using a commercial Miniprep kit). Details can be found under Implementation.



  • mCyPet <partinfo>BBa_K422014</partinfo> has a distinct fluorescence spectra. Measured at an excitation wavelength of 425 nm with a Fluorescein High Precision Monochromator, maximum emission is detected at 482 nm. More information and figures are availabe under Implementation.

Favourite parts

The Archaeal light receptor <partinfo>BBa_K422001</partinfo> comprising of a fusion of the Natronobacterium pharaonis Np seven-transmembrane retinylidene photoreceptor sensory rhodopsins II NpSRII and their cognate transducer HtrII to the cytoplasmic domain of the chemotaxis transducer EcTsr of Escherichia coli is definitely our favourite part. The gene has been codon optimized for expression in E. lemming and subcloned into an IPTG inducible vector. Check out our movies showing the implementation of this chimeric receptor.

The other favourite parts are the key element of the E. lemming: The light induced dimerizing domains phytochrome interacting factor Pif3 <partinfo>BBa_K422012</partinfo> and the photoreceptor PhyB <partinfo>BBa_K422013</partinfo>! Although not present as a fusion construct with a Che-protein and an Anchor-protein yet, these two phytochromes will make the light-switch possible. We are very excited to test this interaction in the short future.

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