Team:TU Eindhoven/Microfluidics/Introduction

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                   <h2>Microfluidics: Introduction</h2>
                   <h2>Microfluidics: Introduction</h2>
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                   <p>A substantial part of the TU Eindhoven iGEM 2014 Project is Microfluidics. Microfluidics is a technique that comprises various fields of engineering. This technique operates on a microscale and thus uses small volumes. For the encapsulation of bacterial cells – as is the case for our iGEM Project – droplet-based microfluidics is used. As extensively elaborated in the Project Overview Page, the engineered bacteria must be brought in the proximity of PEG polymers. The ultimate goal is to verify the intended function of a cell encapsulation device. Theoretically three processes have to be accomplished:</p>
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                   <p>A substantial part of the TU Eindhoven iGEM 2014 Project is Microfluidics. Microfluidics is a technique that comprises various fields of engineering. This technique operates on a microscale and thus uses small volumes. For the encapsulation of bacterial cells – as is the case for our iGEM Project – droplet-based microfluidics is used. As extensively elaborated in the <a href="https://2014.igem.org/Team:TU_Eindhoven/Overview">General Overview Page</a>, the engineered bacteria must be brought in the proximity of PEG polymers. The ultimate goal is to verify the intended function of a cell encapsulation device. Theoretically three processes have to be accomplished:</p>
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The main advantage of these reactions is that they can be applied to in vivo incorporation of unnatural amino acids containing azides or alkynes during translation and expression of proteins. In this way, in theory all bio-orthogonal molecules, such as fluorescent or chemical reporters, can be ‘clicked’ on proteins. (Meldal & Tornoe, 2008)
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Before these processes can be performed, certain questions have to be answered and certain research has to be done. For instance, how is it possible to form droplets? What is the optimal method to recollect the content of the droplets? And numerous of other questions that need answering for instance the required flow speed of the water and oil phase and the viscosities of the phases.
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<h3 id='Staudinger'>Staudinger Ligation</h3>
 
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                  <p>The Staudinger ligation is a modification of the classical Staudinger reaction, which implies the use of phosphines and azides. <NOBR>(<a href='#Fig1'>Figure 1</a>)</NOBR> The Staudinger ligation is arguably the first bio-orthogonal reaction and involves two fully abiotic functional groups and takes place at ambient temperature, in water and at neutral pH. (Baskin & Bertozzi, 2007) However, Vugts et al. showed that the Staudinger ligation is not bio-orthogonal and efficient enough in mice and that the slow reaction kinetics also severely restrict the applicability of the Staudinger ligation in vivo. (Vugts, et al., 2011)</p>
 
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/1/1d/TU_Eindhoven_Staudinger_ligation.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. A schematic overview of the Staudinger ligation.</p>
 
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<h3 id='Copper'>Copper Catalysed [3+2] Azide-Alkyne Cycloaddition (CuAAC)</h3>
 
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                  <p>The reaction often is referred to as ‘the click reaction’, is the extremely selective copper catalysed [3+2] cycloaddition of an azide and an alkyne (CuAAC). (<a href='#Fig2'>Figure 2</a>) CuAAC is a variant of the classical [3+2] cycloaddition discovered by Huisgen, in which copper serves as catalyst, creating a reaction that proceeds rapidly at room temperature. The copper is also the biggest disadvantage of this reaction, since this heavy metal is toxic to cells and organisms. (Meldal & Tornoe, 2008) Nowadays, several suitable Cu(I) ligands are available that minimize the cytotoxicity while maintaining or even further increasing the rate of CuAAC. Therefore, CuAAC has become a very feasible option for in vivo studies. (Yang, Li, & Chen, 2014)</p>
 
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/c/c2/TU_Eindhoven_CuAAC.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 2. A schematic overview of CuAAC.</p>
 
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<h3 id='Strain'>Strain Promoted [3+2] Azide-Alkyne Cycloaddition (SPAAC)</h3>
 
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                  <p>In order to avoid the disadvantages of copper, a copper free version of CuAAC called SPAAC has been designed. In SPAAC, instead of copper, ring strain is used to activate the alkyne. (<a href='#Fig3'>Figure 3</a>) Agard et al. showed that an appropriately derivatized cyclooctyne can efficiently and selectively react with azides in biological mixtures. (Agard, Prescher, & Bertozzi, 2004)</p>
 
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<img id='Fig3' src="https://static.igem.org/mediawiki/2014/3/3f/TU_Eindhoven_SPAAC.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 3. A schematic overview of SPAAC.</p>
 
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<p>Because of the efficiency and selectivity of SPAAC and the fact that no further reagents have to be added to the reaction mixture, SPAAC was chosen to further explore as a platform to functionalize bacterial membranes. Currently, the fastest cyclooctyne known is BARAC. However, this cyclooctyne has shown to be susceptible to Michael addition by thiols , which is not favourable when applying the system into a protein, since proteins contain lots of thiols (cysteines). Dibenzocycloocytne (DBCO) has a lower rate constant, but does not show susceptibility to Michael addition. (Debets, et al., 2014) Furthermore, there are already several DBCO-functionalized polymers and fluorophores commercially available. Therefore, the SPAAC reaction between DBCO-functionalized molecules, which will be reacted azide-functionalized amino acids that have been incorporated into the membrane of <i>E. coli</i>. (<a href='#Fig4'>Figure 4</a>)</p>
 
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<img id='Fig4' src="https://static.igem.org/mediawiki/2014/5/57/TU_Eindhoven_SPAAC_-_DBCO.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 4. A schematic overview of the bio-orthogonal click chemistry that will be conducted in our project: SPAAC using a DBCO-functionalized polymer A and an azide-functionalized amino acid B.</p>
 
<h4>Bibliography</h4>
<h4>Bibliography</h4>

Revision as of 11:00, 9 October 2014

iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Microfluidics: Introduction

A substantial part of the TU Eindhoven iGEM 2014 Project is Microfluidics. Microfluidics is a technique that comprises various fields of engineering. This technique operates on a microscale and thus uses small volumes. For the encapsulation of bacterial cells – as is the case for our iGEM Project – droplet-based microfluidics is used. As extensively elaborated in the General Overview Page, the engineered bacteria must be brought in the proximity of PEG polymers. The ultimate goal is to verify the intended function of a cell encapsulation device. Theoretically three processes have to be accomplished:

  1. Combining a PEG solution and a bacterial culture with our engineered E. coli bacteria. These are the essential substances to perform the click reaction. After combining, this is called the Water Phase.

  2. The Water Phase has to be dispersed with an Oil Phase into droplets. This oil phase contains a fluorosurfactant in order to prevent aggregation and agglomeration of the droplets. It is crucial to form droplets with one cell in each droplet, since it will assure that each bacterial cell is encapsulated correctly – possible formation of a film is hereby avoided.

  3. The result of the previous two processes is a droplet with PEG and engineered E. coli bacteria inside an oil phase. With a microfluidic feature (bumpy mixer) the droplet can be stirred. The droplets are collected in a chamber where the click reaction is initiated with the use of UV light.

Before these processes can be performed, certain questions have to be answered and certain research has to be done. For instance, how is it possible to form droplets? What is the optimal method to recollect the content of the droplets? And numerous of other questions that need answering for instance the required flow speed of the water and oil phase and the viscosities of the phases.

Bibliography

Agard, N. J., Prescher, J. A., & Bertozzi, C. R. (2004). A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. Journal of the American Chemical Society, 126, 15046-15047.

Baskin, J. M., & Bertozzi, C. R. (2007). Bioorthogonal click chemistry: covalent labeling in living systems. QSAR & Combinatorial Science, 26(11-12), 1211 - 1219.

Debets, M. F., Prins, J. S., Merkx, D., van Berkel, S. S., van Delft, F. L., van Hest, J. C., & Rutjes, F. P. (2014). Synthesis of DIBAC analogues with excellent SPAAC rate constants. Organic & Biomolecular Chemistry, 12, 5031-5037.

Meldal, M., & Tornoe, C. W. (2008). Cu-catalyzed azide-alkyne cycloaddition. Chemical Reviews, 108, 2952-3015.

Vugts, D. J., Vervoort, A., Stigter-van Walsum, M., Visser, G. W., Robillard, M. S., Versteegen, R. M., . . . van Dongen, G. A. (2011). Synthesis of phosphine and antibody-azide probes for in vivo Staudinger ligation in a pretargeted imaging and therapy approach. Bioconjugate Chemistry, 22, 2072-2081.

Yang, M., Li, J., & Chen, P. R. (2014). Transition metal-mediated bioorthogonal protein chemistry in living cells. Chemical Society Reviews, 43(18), 6475-6660.

iGEM Team TU Eindhoven 2014

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