This work was supported by the Austrian Science Fund (FWF Project W1224 - Doctoral Program on Biomolecular Technology of Proteins - BioToP and FWF Project ESP 465-B), the Federal Ministry for Digital and Economic Affairs of Austria, the National Foundation for Research, Technology and Development of Austria to the Christian Doppler Research Association (Christian Doppler Laboratory for Next Generation CAR T Cells), and by private donations to the St. Anna Children&#39;s Cancer Research Institute (Vienna, Austria). E.S. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the St. Anna Children&#39;s Cancer Research Institute. Figures were created with BioRender.com.

Optimizing Protein Functions Through Yeast Surface Display Techniques

M.W.T. receives funding from Miltenyi Biotec. All authors are inventors on patent applications for technologies and engineered proteins that were developed by using yeast surface display.

Yeast surface display has evolved as one of the key methods used in protein engineering. Although it is commonly employed for the engineering of affinity1,18,40,41, expression/stability24,27,42,43 and activity28,44, further uses like epitope mapping45,46 or characterization of the individual mutants on the surface of yeast cells9 are possible as well. In this protocol, we provide the basic steps for starting a yeast surface display selection campaign, including the selection with magnetic beads and by flow cytometric sorting as well as diversification of the yeast library by epPCR for affinity maturation. 
One essential requirement for conventional yeast surface display selections is the availability of soluble protein of sufficient quality. Starting with a well-folded target protein with high purity and a defined oligomerization state (i.e., monomeric protein should only be present as monomer) provides the highest success rate to select for a protein variant binding to the target antigen with high affinity. An alternative for difficult-to-express target proteins is cell-based selections, which present a reasonable strategy to circumvent this limitation47. However, yeast surface display offers many advantages, such as the possibility of characterizing resulting protein variants directly on the surface of yeast without the necessity to perform laborious and time-intensive cloning, expression in a soluble format, and protein purification. Both the affinity and the stability of the variants can be analyzed directly on the yeast surface9. 
In this protocol, we show how the G4 library of protein variants, more specifically of the 10th type III domain of human fibronectin, was selected for binding to the antigen hRBP4 in the presence of the small molecule A1120. The combination of bead selections and flow cytometric sorting yielded an enrichment of variants, which showed an increased binding to the target antigen throughout selection rounds (Figure 2B). We showed that using lower concentrations of antigen enables the selection of high-affinity protein variants (Figure 2C). Typically, affinities that can be achieved with yeast display selections are in the nanomolar or even picomolar range18. The final affinities depend on the target antigen, the number of selection rounds and affinity maturation, the binding scaffold used, and the applied gating strategy. The characterization of individual protein variants is not covered in this protocol but is explained in detail in our previous work9. Although yeast display was originally employed for the engineering of antibody fragments such as scFvs1,40, the method has been widely used for non-antibody-based proteins as well10. 
To sum up, yeast surface display is a powerful protein engineering tool that enables the generation of protein variants with novel or improved properties, such as binding to almost any target protein and/or increased stability.

Yeast surface display is one of the key technologies in the field of protein engineering. It enables the selection of protein variants with desired properties such as improved affinity or stability. First introduced in 19971, it is one of the most commonly used display technologies besides phage display2,3, ribosome display4, and mammalian cell display5,6,7. The protein of interest (POI) is displayed on the surface of yeast cells by fusing it to anchor proteins. A range of different anchor proteins is available, and most commonly, the POI is fused to the C-terminus of the yeast agglutinin mating protein Aga2p1,8. Additionally, the POI is typically flanked by two tags, such as a hemagglutinin tag (HA-tag) and c-myc tag, which enables detection of the display level by using fluorescently labeled antibodies and flow cytometry (Figure 1A). Typical yeast selection campaigns involve a combination of magnetic bead selections and flow cytometric sorting. The bead selections enable the handling of large cell numbers and enrichment of protein variants binding to the target antigen also with low affinities since multivalent interactions with the antigen-loaded beads lead to avidity effects and, therefore, prevent the loss of low-affinity variants (Figure 1B). Flow cytometric analysis and selection offer the advantage of visualizing the binding of the displayed POI variants to the labeled antigen. Consequently, the binding populations can be sorted and cultivated, leading to the enrichment of protein variants with desired characteristics throughout several sorting rounds. Moreover, additional rounds of random mutagenesis can be performed to further increase the diversity and, hence, the likelihood of finding additional mutations that contribute to the affinity and/or stability of the protein.
Yeast surface display presents certain advantages, such as (a) eukaryotic expression machinery, enabling oxidative protein folding as well as eukaryotic post-translational modifications (such as N-glycosylation), (b) expression normalization due to the detection of the two peptide tags flanking the protein, (c) visual inspection of the selection progress by flow cytometry (e.g., percentage of binding cells and binding intensity) and (d) the possibility of analyzing individual protein mutants on yeast (e.g., analyzing thermostability as well as affinity), presenting a time-saving alternative to laborious protein expression and purification9. In fact, both affinities (KD values) as well as stabilities (T50 values) of yeast surface displayed proteins have shown good correlations with data obtained using biophysical methods and soluble proteins9,10,11,12. Yeast surface display has been employed for the engineering of a variety of proteins, e.g., antibody fragments13,14,15,16, the 10th type III fibronectin domain17,18, rcSso7d19,20, or knottins21. Similarly, extensive research has been undertaken to optimize yeast library designs by altering the randomized positions as well as amino acid codon usage17,22,23. Yeast surface display has been proven successful for the engineering of stability14,15,24,25, affinity18,26,27, enzymatic activity28,29,30,31, and protein expression32. Additionally, more sophisticated applications like conditional binding in the presence or absence of a small molecule were accomplished using yeast surface display20.
In this protocol, we describe all essential steps for a selection campaign with yeast surface display with the example of the G4 library (based on the 10th type III fibronectin domain, Fn3) selected against the antigen human retinol-binding protein 4 (hRBP4) in the presence of the small molecule A112020. This selection was conducted to yield a protein-protein interaction which is dependent on a small molecule that can be used as a molecular switch. Of note, while alternative approaches are possible with yeast surface display, typical yeast selections usually aim for binding to a target antigen without any previous binding affinity. We cover all steps of a yeast selection campaign, involving the cultivation of a yeast library, bead selections, flow cytometric sorting, and affinity maturation by error-prone PCR (epPCR). Therefore, this protocol complements previous yeast surface display protocols33,34 and can be used as a basis for yeast surface display selections (Figure 1) with any given yeast library and target antigen of choice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66994/66994fig1v5.jpg" /> 
Figure 1: Principle of yeast surface display and a typical workflow for yeast surface display selections. (A) The POI is cloned into a yeast surface display vector and typically flanked by an N-terminal HA- and a C-terminal c-myc-tag. The construct is fused to the yeast mating protein Aga2p for display on the surface. The protein depicted is the engineered binder &#34;RS3&#34; from PDB ID: 6QBA20. (B) Flowchart illustrating a typical workflow for yeast surface display selection campaigns, which combine enrichment of protein variants with desired properties by bead selections and flow cytometric sorting, as well as epPCR for affinity maturation. <a href="https://www.jove.com/files/ftp_upload/66994/66994fig1largev5.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10-beta electrocompetent E. coli&#160;</td>
			<td>NEB</td>
			<td>C3020K</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar-Agar, Kobe I</td>
			<td>Carl Roth</td>
			<td>5210.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Carl Roth</td>
			<td>K029.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-c-myc antibody, clone 9E10, AF488</td>
			<td>Invitrogen</td>
			<td>MA1-980-A488 (Thermo Fisher)</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-c-myc antibody, clone 9E10, AF647</td>
			<td>Invitrogen</td>
			<td>MA1-980-A647 (Thermo Fisher)</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HA antibody, clone 16B12, AF488</td>
			<td>BioLegend</td>
			<td>901509 (Biozym)</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HA antibody, clone 16B12, AF647</td>
			<td>BioLegend</td>
			<td>682404 (Biozym)</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>NEB</td>
			<td>R3136S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, cold ethanol fraction</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Galactose</td>
			<td>Carl Roth</td>
			<td>4987.2</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco yeast nitrogen base</td>
			<td>Becton Dickinson (BD)&#160;</td>
			<td>291940</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-Sodium hydrogen phosphate heptahydrate</td>
			<td>Carl Roth</td>
			<td>X987.3</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Carl Roth</td>
			<td>6213.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffered saline (10x)</td>
			<td>Thermo Scientific</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Biotin Binder</td>
			<td>Invitrogen</td>
			<td>11047 (Fisher Scientific)</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Thermo Fisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>EBY100</td>
			<td>ATCC</td>
			<td>MYA-4941</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvette 1 mm (for E.coli)</td>
			<td>VWR</td>
			<td>732-1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvette 2 mm (for yeast)</td>
			<td>VWR</td>
			<td>732-1136</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>MERCK</td>
			<td>1070172511</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneMorph II Random Mutagenesis Kit</td>
			<td>Agilent Technologies</td>
			<td>200550</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Bacto Casamino Acids</td>
			<td>Becton Dickinson (BD)</td>
			<td>223120</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem</td>
			<td>131339.1211</td>
			<td></td>
		</tr>
		<tr>
			<td>LE agarose&#160;</td>
			<td>Biozym</td>
			<td>840004</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium acetate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td></td>
		</tr>
		<tr>
			<td>Monarch DNA Gel Extraction Kit</td>
			<td>NEB</td>
			<td>T1020S</td>
			<td></td>
		</tr>
		<tr>
			<td>Monarch PCR &#38; DNA Cleanup Kit</td>
			<td>NEB</td>
			<td>T1030S</td>
			<td></td>
		</tr>
		<tr>
			<td>Multifuge 1S-R</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NheI-HF</td>
			<td>NEB</td>
			<td>R3131S</td>
			<td></td>
		</tr>
		<tr>
			<td>Outgrowth medium</td>
			<td>NEB</td>
			<td>B9035S</td>
			<td></td>
		</tr>
		<tr>
			<td>pCTCON2</td>
			<td>Addgene</td>
			<td>#41843</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Penta-His antibody, AF488</td>
			<td>Qiagen</td>
			<td>35310</td>
			<td></td>
		</tr>
		<tr>
			<td>Penta-His antibody, AF647</td>
			<td>Qiagen</td>
			<td>35370</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone ex casein tryptically digested</td>
			<td>Carl Roth</td>
			<td>8986.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 High-Fidelity DNA Polymerase&#160;</td>
			<td>NEB</td>
			<td>M0491S</td>
			<td></td>
		</tr>
		<tr>
			<td>SaII-HF</td>
			<td>NEB</td>
			<td>R3138S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S8750-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>3957.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>Carl Roth</td>
			<td>K300.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Steritop threaded bottle top filter</td>
			<td>MERCK</td>
			<td>S2GPT01RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin, AF488</td>
			<td>Invitrogen</td>
			<td>S32354 (Thermo Fisher)</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin, AF647</td>
			<td>Invitrogen</td>
			<td>S32357 (Thermo Fisher)</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Sodium citrate dihydrate</td>
			<td>Carl Roth</td>
			<td>4088.1</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer</td>
			<td>Agilent</td>
			<td>8453</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract, micro-granulated</td>
			<td>Carl Roth</td>
			<td>2904.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoprep Yeast Plasmid Miniprep II</td>
			<td>Zymo Research</td>
			<td>D2004&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

protein engineering by yeast surface display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

The G4 library was selected against the antigen hRBP4 bound to the small molecule drug A1120. The staining of the libraries for flow cytometric sorting was performed as described in Method 6, and the applied gating strategy is shown in Figure 2A. An initial gate included all cells based on cell morphology, and the second gate (histogram of FSC-Width) showed a stringent gating strategy that was applied to select single cells and remove cell aggregates. The third and final gate showed the display of protein variants (x-axis) versus antigen binding (y-axis). Yeast cells showing both display and binding signals were sorted. Importantly, the sorting gate was set in a stringent way to enrich binding domains with high binding signal and thus high affinity. This stringent selection yielded an enrichment of displaying yeast cells that specifically bind to the target antigen throughout the selection campaign (Figure 2B). In later flow cytometric sorting rounds, the antigen concentration was decreased 10-fold (from 100 nM to 10 nM). Therefore, the overall binding signal was reduced, and only binders with a high affinity were still detectable and sorted (Figure 2C).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66994/66994fig2v5.jpg" /> 
Figure 2: Representative results from a yeast surface display selection of the Fn3-based G4 library for binding to the antigen (hRBP4 in the presence of A1120). (A) The general gating strategy for sorting of yeast libraries. The first gate (FSC vs. SSC) is to select all yeast cells and exclude scatter events; the second gate (histogram of FSC-W) aims to remove cell aggregates and only select single yeast cells. The third gate plots the surface display level (detection of the HA- or c-myc-tag) vs. binding to the antigen (here hRBP4 in the presence of 5 &#181;M A1120, detected by anti-His antibody). The library was additionally stained with secondary antibodies only (without antigen), where no antigen binding is expected. Sorted cells are highlighted in blue. (B) Evolution of the G4 library throughout 3 rounds of flow cytometric sorting. Enrichment of the binding population can be observed with each selection round. (C) The use of lower antigen concentrations enables the selection of protein variants with a higher affinity towards the target antigen. Upon reduction of the antigen concentration (here hRBP4) by 10-fold, different diagonals appear, indicating the presence of clones with higher (sorted cells, blue) or lower affinity. <a href="https://www.jove.com/files/ftp_upload/66994/66994fig2largev5.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Protein Engineering by Yeast Surface Display

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

protein-engineering-by-yeast-surface-display

Research

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JoVE Journal

Bioengineering

720 Views.  BOKU University. This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Video: Protein Engineering by Yeast Surface Display

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.