Team:Edinburgh/Test

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#redirect [[Team:Edinburgh]]
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<p class="h1">Data Overview</p> <!-- see https://igem.org/Sample_Data_Page for example page. -->
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This page provides an overview of the purely biological aspects of our feasibility study, and links to the relevant Parts Registry pages.
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==Cell Surface Display System==
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(Further details are at the dedicated [[Team:Edinburgh/Cell Display | Cell Surface Display]] page.)
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This system aims at achieving synergy between the enzymes by displaying them at high copy number on the cell's outer membrane. <span class="hardword" id="inp">Ice Nucleation Protein</span> is used as a carrier for display of the enzymes; it carries them to the outer membrane.
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===Schematic diagram===
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[[Image:Edinburgh-Data-Cell-Display.png|thumb|center|710px|<br>The completed system should contain:<br>
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&nbsp; A '''promoter''' (<partinfo>BBa_K523000</partinfo>) controlling:<br>
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&nbsp; &nbsp; an '''INP&mdash;Endoglucanase''' fusion (<partinfo>BBa_K523008</partinfo> + <partinfo>BBa_K523011</partinfo>)<br>
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&nbsp; &nbsp; an '''INP&mdash;&beta;-glucosidase''' fusion (<partinfo>BBa_K523008</partinfo> + <partinfo>BBa_K523004</partinfo>)<br>
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&nbsp; &nbsp; an '''INP&mdash;Exoglucanase''' fusion (<partinfo>BBa_K523008</partinfo> + <partinfo>BBa_K523009</partinfo>)<br><br>
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'''Ribosome Binding Sites''' are indicated as green ovals.<br><br>
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Cellulose degradation is shown at the bottom. In reality, tens of thousands of enzymes will cover the outer membrane in random places.<br><br>A test system to prove that <partinfo>BBa_K523008</partinfo> can be used to carry proteins to the outer membrane uses a fusion of INP to '''Yellow Fluorescent Protein (YFP)''' instead.]]
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===Progress===
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We have:
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* Shown that INP (<partinfo>BBa_K523008</partinfo>, based on <partinfo>BBa_K265008</partinfo>) can be used to carry proteins to the cell membrane, by constructing <partinfo>BBa_K523013</partinfo>.
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* Made and tested a new &beta;-glucosidase ''(bglX)'' BioBrick, <partinfo>BBa_K523002</partinfo>.
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* Made ''bglX'' into a format compatible with our BioSandwich assembly protocol (<partinfo>BBa_K523004</partinfo>).
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* Made new versions of other cellulases, in a format compatible with the BioSandwich protocol.
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* Tested an old exoglucanase gene, <partinfo>BBa_K118022</partinfo>.
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* Made fusions of INP to ''bglX'' (&beta;-glucosidase) and ''cex'' (exoglucanase).
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==Phage Display System==
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(Further details are at the dedicated [[Team:Edinburgh/Phage Display | Phage Display]] page.)
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This system aims at achieving synergy between the enzymes by displaying them on an <span class="hardword" id="m13">M13</span> <span class="hardword" id="phage">phage</span>. The major coat protein <span class="hardword" id="p8">pVIII</span> is used as a carrier for display of the enzymes; it incorporates them into the phage.
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===Schematic diagram===
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[[Image:Edinburgh-Data-Phage-Display.png|thumb|center|710px|<br>The completed system should contain:<br>
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&nbsp; A '''promoter''' (<partinfo>BBa_K523000</partinfo>) controlling:<br>
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&nbsp; &nbsp; an '''Endoglucanase&mdash;pVIII''' fusion<br>
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&nbsp; &nbsp; a '''&beta;-glucosidase&mdash;pVIII''' fusion<br>
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&nbsp; &nbsp; an '''Exoglucanase&mdash;pVIII''' fusion<br><br>
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'''Ribosome Binding Sites''' are indicated as green ovals. '''"Signal"''' means a periplasmic signal sequence, directing the protein to the periplasm to be assembled into the phage.<br><br>A test system uses a fusion of pVIII to ''E. coli'' amylase '''MalS''' instead.]]
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===Progress===
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We have:
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* Made and tested a new amylase ''(malS)'' BioBrick, <partinfo>BBa_K523001</partinfo>.
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* Attempted to fuse it to the major coat protein pVIII.
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==BioSandwich==
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BioSandwich (<nowiki>RFC 81</nowiki>) is a new assembly protocol that we used.
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Parts made using BioSandwich that we believe are correct (subject to final verification sequencing) include:
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* <partinfo>BBa_K523013</partinfo>: PlacLacZ-INP-YFP
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* <partinfo>BBa_K523019</partinfo>: PlacLacZ-INP-cex
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* <partinfo>BBa_K523020</partinfo>: PlacLacZ-INP-bglX
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* <partinfo>BBa_K523022</partinfo>: PlacLacZ-crtEIB
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==Favourite BioBricks==
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(A complete list of parts made during the project is found at the dedicated [[Team:Edinburgh/Parts | Parts]] page.)
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; '''<partinfo>BBa_K523000</partinfo>: Plac + lacZ; with 5' BglII site'''
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: Intended as a cloning vector (when present in pSB1C3), this part allows Blue/White selection of newly PCR'ed BioBricks. The PCR primers required are shorter than would be required using the standard method. It encodes LacZ&alpha;; new BioBricks that have successfully replaced the part will be white on plates containing IPTG and Xgal. Parts created in this way will also be compatible with the BioSandwich assembly protocol. We proved this part worked by using it to create several other BioBricks.
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; '''<partinfo>BBa_K523006</partinfo>: Plac + malS'''
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: ''malS'' is a <span class="hardword" id="periplasm">periplasmic</span> ''E. coli'' <span class="hardword" id="amylase">amylase</span> gene. It ought to be usable for a fusion to INP or pVIII. But to test its normal activity, we placed it under the control of the lac promoter in a high copy number plasmid. We found evidence of <span class="hardword" id="starch">starch</span> degrading activity.
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; '''<partinfo>BBa_K523013</partinfo>: Plac + INP-EYFP'''
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: A fusion of Ice Nucleation Protein and Enhanced Yellow Fluorescent Protein under the control of the Lac promoter. Fluoresces yellow under blue light. May be localised to the outer membrane.
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; '''<partinfo>BBa_K523014</partinfo>: Plac + bglX'''
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: ''bglX'' is a <span class="hardword" id="cryptic">cryptic</span> &beta;-glucosidase gene from ''E. coli''. It ought to be usable for a fusion to INP or pVIII. But to test its normal activity, we placed it under the control of the lac promoter in a high copy number plasmid. We found it to be capable of degrading the <span class="hardword" id="cellobiose">cellobiose</span> analog, MUG.
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==Other BioBricks analysed==
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(More details of this are found at the dedicated [[Team:Edinburgh/Collaboration | Collaboration]] page. The information has also been added to the parts' "Experience" sections.)
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; '''[http://partsregistry.org/Part:BBa_K118022:Experience BBa_K118022]: ''C. fimi'' exoglucanase'''
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: At the same time as we tested ''bglX'' for activity, we also tested this old part's ability to degrade the cellobiose analog MUG (weak ability) and the cellulose analog MUC (strong ability).
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; '''[http://partsregistry.org/Part:BBa_K415151:Experience BBa_K415151]: p8-GR1'''
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: Supposed to encode a fusion of the pVIII protein to a GR1 zipper. It actually encodes part of the ''E. coli'' aminopeptidase N gene.
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; '''[http://partsregistry.org/Part:BBa_K392008:Experience BBa_K392008]: ''C. fimi'' &beta;-glucosidase'''
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: We discovered that this part almost certainly starts its coding sequence at the second ATG present, not the first. This is highly relevant information for anyone trying to tune its expression via use of custom Ribosome Binding Sites.
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Latest revision as of 22:41, 21 September 2011

  1. redirect Team:Edinburgh
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