Difference between revisions of "Team:LMU-TUM Munich/Results"

(The sub-projects of biotINK and their achievements)
(The sub-projects of biotINK and their achievements)
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Design BioBrick Part Design] ==
 
== [https://2016.igem.org/Team:LMU-TUM_Munich/Design BioBrick Part Design] ==
 
[[File:Muc16_Sticker_Design_001.png |right|200px]]
 
[[File:Muc16_Sticker_Design_001.png |right|200px]]
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As does every sophisticated SynBio project, we designed a plethora of BioBrick constructs and devices in the course of our project. Most prominent among these are the modular transmembrane proteins we conceptualized, since they are integral to the functionality of our bioprinting system. Basing our workflow on the standard RFC[10] and RFC[25] assembly standards, we assembled the same intracellular and transmembrane domains, namely mRuby3 and the EGFR transmembrane domain, with different extracellular domains, among them single-chain avidin, monomeric avidin, and NanoLuc fused with an N-terminal Avi-tag for ''in vivo'' biotinylation by the co-expressed biotin ligase BirA. We coupled these constructs with a trafficking signal for membrane integration. Including further elements to stabilize these receptors offers advantages over previously assembled BioBricks, and planning our constructs with multiple options for detection provided us ample opportunity to extensively characterize the outcome. Apart from these eukaryotic designs, we drafted an autotransporter construct for bacterial surface display.
 
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Proteins Production, purification & conjugation of recombinant proteins] ==
 
== [https://2016.igem.org/Team:LMU-TUM_Munich/Proteins Production, purification & conjugation of recombinant proteins] ==
 
[[File:Muc16_Sticker_proteins_001.png |right|200px]]
 
[[File:Muc16_Sticker_proteins_001.png |right|200px]]
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As the second constituent of our two-component polymerization system next to the membrane proteins, we required large amounts of protein, either biotin-binding or bearing chemically introduced biotin depending on the receptor in question. For biotin-binding proteins, we produced and purified the avidin variants wt streptavidin; traptavidin,a variant with higher affinity toward biotin and enhanced thermal stability; streptactin, a variant that binds an oligopeptide sequence termed strep-tag; and enhanced monomeric avidin, which does not assemble into tetramers and is thus monovalent. wt Streptavidin was produced both in the dimensions of shaker flasks and a fermenter, and purified according to an established protocol. As a scaffold for chemical biotinylation, we produced and purified PAS-lysine and GFP. We extensively characterized all proteins as well as the biotinylated forms of the latter group plus BSA by ESI-TOF, SDS-PAGE, and IEF; moreover, we determined the affinity of streptavidin variants ''via'' fluorescence titration and their thermal stability using CD spectra. We produced all target proteins in good purity and successfully biotinylated PAS-lysine, GFP, and BSA.
 
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Localization A fully functional cross-kingdom membrane lokalization toolbos] ==
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Receptors A fully functional cross-kingdom membrane localization toolbox] ==
 
[[File:Muc16_Sticker_Receptors_003.png |right|200px]]
 
[[File:Muc16_Sticker_Receptors_003.png |right|200px]]
 +
Based on our modular membrane protein design, we evaluated the functionality of different signal peptide in shuttling the protein into the membrane. Apart from taking the BioBrick EGFR signal peptide, we designed two further signal peptides with increased translational efficiency, incorporating items such as the Kozak sequence. These three signal peptides were analyzed ''in silico'' as well as experimentally using a luciferase secretion assay. The BM40 signal, one of our self-designed constructs, was most efficient and thus incorporated into our other devices. Additionally, we incorporated three reporter modules (mRuby3, A3C5 epitope tag and strep-tag II) into the receptors. The finished constructs were used to generate semi-stablly transfected and stable cell lines. Protein localization was confirmed ''via'' fluorescence and high-resolution fluorescence microscopy, including characterization of a trafficking signal for increased membrane localization, and the recombinant protein was quantified by FACS and Western blot. mRNA expression levels were detected by RT-q-PCR. For visualization, aggregation of functionalized cells with Strep beads was subjected to SEM. The bacterial autotransporter was characterized and found to successfully shuttle small protein domains to the extracellular surface.
 
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Linkerchemistry Chemical engineering of amino-reactive biotinylation compounds] ==
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Linkerchemistry Chemical synthesis of amino-reactive biotinylation compounds] ==
 
[[File:Muc16_Sticker_Chemistry_001.png |right|200px]]
 
[[File:Muc16_Sticker_Chemistry_001.png |right|200px]]
 +
Apart from the standard biotin-NHS, several other derivatives spacing biotin and NHS apart by an amino acid residue were synthesized. These derivatives were used in the chemical biotinylation of BSA in order to assess their relative stability toward cleavage by the plasma protein biotinidase. The assay principle is based on a colorimetric reaction and was developed by ourselves. Compared to biotinylation ''via'' regular biotin-NHS, the derivative incorporating a spacer aminocaproic acid is cleaved somewhat faster, allowing to dissolve a cell mesh more quickly, and the spacer valine resulted in a drastically slowed hydrolysis rate.
 
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Proof Proof-of-Prinziple for the Avidin-Biotin "polymerization"] ==
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Proof Proof of principle for the avidin-biotin "polymerization"] ==
 
[[File:Muc16_Sticker_Proof_001.png |right|200px]]
 
[[File:Muc16_Sticker_Proof_001.png |right|200px]]
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Hypoxia A vascularization inducer based on a hypoxia sensitive promoter] ==
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Hypoxia A vascularization inducer based on a hypoxia-sensitive promoter] ==
 
[[File:Muc16_Sticker_Hypoxia_001.png |right|200px|]]
 
[[File:Muc16_Sticker_Hypoxia_001.png |right|200px|]]
 
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Safety Saftety evaluation of our project and a Kill-switch] ==
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== [https://2016.igem.org/Team:LMU-TUM_Munich/Safety Safety evaluation of our project and the kill-switch] ==
 
[[File:Muc16_Sticker_Safety_001.png |right|200px|]]
 
[[File:Muc16_Sticker_Safety_001.png |right|200px|]]
 
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Revision as of 03:09, 20 October 2016

Muc16 BioBrick summary 001.png

The sub-projects of biotINK and their achievements

BioBrick Part Design

Muc16 Sticker Design 001.png

As does every sophisticated SynBio project, we designed a plethora of BioBrick constructs and devices in the course of our project. Most prominent among these are the modular transmembrane proteins we conceptualized, since they are integral to the functionality of our bioprinting system. Basing our workflow on the standard RFC[10] and RFC[25] assembly standards, we assembled the same intracellular and transmembrane domains, namely mRuby3 and the EGFR transmembrane domain, with different extracellular domains, among them single-chain avidin, monomeric avidin, and NanoLuc fused with an N-terminal Avi-tag for in vivo biotinylation by the co-expressed biotin ligase BirA. We coupled these constructs with a trafficking signal for membrane integration. Including further elements to stabilize these receptors offers advantages over previously assembled BioBricks, and planning our constructs with multiple options for detection provided us ample opportunity to extensively characterize the outcome. Apart from these eukaryotic designs, we drafted an autotransporter construct for bacterial surface display.


Production, purification & conjugation of recombinant proteins

Muc16 Sticker proteins 001.png

As the second constituent of our two-component polymerization system next to the membrane proteins, we required large amounts of protein, either biotin-binding or bearing chemically introduced biotin depending on the receptor in question. For biotin-binding proteins, we produced and purified the avidin variants wt streptavidin; traptavidin,a variant with higher affinity toward biotin and enhanced thermal stability; streptactin, a variant that binds an oligopeptide sequence termed strep-tag; and enhanced monomeric avidin, which does not assemble into tetramers and is thus monovalent. wt Streptavidin was produced both in the dimensions of shaker flasks and a fermenter, and purified according to an established protocol. As a scaffold for chemical biotinylation, we produced and purified PAS-lysine and GFP. We extensively characterized all proteins as well as the biotinylated forms of the latter group plus BSA by ESI-TOF, SDS-PAGE, and IEF; moreover, we determined the affinity of streptavidin variants via fluorescence titration and their thermal stability using CD spectra. We produced all target proteins in good purity and successfully biotinylated PAS-lysine, GFP, and BSA.




A fully functional cross-kingdom membrane localization toolbox

Muc16 Sticker Receptors 003.png

Based on our modular membrane protein design, we evaluated the functionality of different signal peptide in shuttling the protein into the membrane. Apart from taking the BioBrick EGFR signal peptide, we designed two further signal peptides with increased translational efficiency, incorporating items such as the Kozak sequence. These three signal peptides were analyzed in silico as well as experimentally using a luciferase secretion assay. The BM40 signal, one of our self-designed constructs, was most efficient and thus incorporated into our other devices. Additionally, we incorporated three reporter modules (mRuby3, A3C5 epitope tag and strep-tag II) into the receptors. The finished constructs were used to generate semi-stablly transfected and stable cell lines. Protein localization was confirmed via fluorescence and high-resolution fluorescence microscopy, including characterization of a trafficking signal for increased membrane localization, and the recombinant protein was quantified by FACS and Western blot. mRNA expression levels were detected by RT-q-PCR. For visualization, aggregation of functionalized cells with Strep beads was subjected to SEM. The bacterial autotransporter was characterized and found to successfully shuttle small protein domains to the extracellular surface.




Chemical synthesis of amino-reactive biotinylation compounds

Muc16 Sticker Chemistry 001.png

Apart from the standard biotin-NHS, several other derivatives spacing biotin and NHS apart by an amino acid residue were synthesized. These derivatives were used in the chemical biotinylation of BSA in order to assess their relative stability toward cleavage by the plasma protein biotinidase. The assay principle is based on a colorimetric reaction and was developed by ourselves. Compared to biotinylation via regular biotin-NHS, the derivative incorporating a spacer aminocaproic acid is cleaved somewhat faster, allowing to dissolve a cell mesh more quickly, and the spacer valine resulted in a drastically slowed hydrolysis rate.




Proof of principle for the avidin-biotin "polymerization"

Muc16 Sticker Proof 001.png






First bioprinting results using the biotINK bioprinter

Muc16 Sticker Demonstrate 001.png






A vascularization inducer based on a hypoxia-sensitive promoter

Muc16 Sticker Hypoxia 001.png






Safety evaluation of our project and the kill-switch

Muc16 Sticker Safety 001.png







InterLab study: Determining reliability of measurements

Muc16 Sticker Interlab 001.png




Entrepreneurship

Muc16 Sticker Entrepreneurship 001.png






Hardware

Muc16 Sticker Hardware 001.png






Software

Muc16 Sticker Software 001.png






Measurement

Muc16 Sticker Measurement 001.png






Model

Muc16 Sticker Model 001.png






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LMU & TUM Munich

Technische Universität MünchenLudwig-Maximilians-Universität München

United team from Munich's universities

Contact us:

Address

iGEM Team TU-Munich
Emil-Erlenmeyer-Forum 5
85354 Freising, Germany