We sincerely thank Thomas Howe and J&#246;rg Carsten for the programming and technical support with the liquid handling systems.

<ol>
	<li>Milani, J., Maleki, G., Valdez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2,+Hydrocolloids+in+Food+Industry.">Ch. 2, Hydrocolloids in Food Industry.</a> Food Industrial Processes - Methods and Equipment. 2, (2012).</li><li>Thibodeau, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+skin+from+environmental+stresses+with+an+exopolysaccharide+formulation.">Protecting the skin from environmental stresses with an exopolysaccharide formulation.</a> Cosmet Toiletries. 120 (12), 81-90 (2005).</li><li>Prajapati, V. D., Jani, G. K., Zala, B. S., Khutliwala, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+emerging+exopolysaccharide+gellan+gum+as+a+novel+polymer.">An insight into the emerging exopolysaccharide gellan gum as a novel polymer.</a> Carbohydr Polym. 93 (2), 670-678 (2013).</li><li>Schmidt, W., Brouwers, H. J. H., Kuehne, H. C., Meng, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+working+mechanism+of+starch+and+diutan+gum+in+cementitious+and+limestone+dispersions+in+presence+of+polycarboxylate+ether+superplasticizers.">The working mechanism of starch and diutan gum in cementitious and limestone dispersions in presence of polycarboxylate ether superplasticizers.</a> Appl. Rheol. 23 (5), 52903 (2013).</li><li>Srinivasan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Polysaccharides+as+Treatment+Agents+for+Wastewater.">Natural Polysaccharides as Treatment Agents for Wastewater.</a> Green Materials for Sustainable Water Remediation and Treatment. , 51-81 (2013).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+exopolysaccharide+screening+platform:+From+strain+cultivation+to+monosaccharide+composition+and+carbohydrate+fingerprinting+in+one+day.">High throughput exopolysaccharide screening platform: From strain cultivation to monosaccharide composition and carbohydrate fingerprinting in one day.</a> Carbohydr Polym. 122, 212-220 (2015).</li><li>Ortega-Morales, B. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+extracellular+polymers+synthesized+by+tropical+intertidal+biofilm+bacteria.">Characterization of extracellular polymers synthesized by tropical intertidal biofilm bacteria.</a> J Appl Microbiol. 102 (1), 254-264 (2007).</li><li>Xu, R., Ma, S., Wang, Y., Liu, L., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+identification+and+statistic+optimization+of+a+novel+exopolysaccharide+producing+Lactobacillus+paracasei+HCT.">Screening identification and statistic optimization of a novel exopolysaccharide producing Lactobacillus paracasei HCT.</a> Afr. J. Microbiol. Res. 4 (9), 783-795 (2010).</li><li>Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+method+for+determination+of+sugars+and+related+substances.">Colorimetric method for determination of sugars and related substances.</a> Anal. Chem. 28 (3), 350-356 (1956).</li><li>Masuko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+analysis+by+a+phenol-sulfuric+acid+method+in+microplate+format.">Carbohydrate analysis by a phenol-sulfuric acid method in microplate format.</a> Anal Biochem. 339 (1), 69-72 (2005).</li><li>Geater, C. W., Fehr, W. R., Wilson, L. A., Robyt, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+more+rapid+method+of+total+sugar+analysis+for+soybean+seed.">A more rapid method of total sugar analysis for soybean seed.</a> Crop Sci. 41 (1), 250-252 (2001).</li><li>Sutherland, I. W., Dumitriu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+16,+Microbial+Exopolysaccharides.">Ch 16, Microbial Exopolysaccharides.</a> Polysaccharides Structural Diversity and Functional Versatility. , 431-457 (2005).</li><li>Wingender, J., Neu, T. R., Flemming, H. C., Wingender, J., Neu, T. R., Flemming, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+Bacterial+Extracellular+Polymeric+Substances?.">What are Bacterial Extracellular Polymeric Substances?.</a> Microbial extracellular polymeric substances: characterization, structure and function. , 258 (1999).</li><li>R&#252;hmann, B., Schmid, J., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+carbohydrate+analysis+via+liquid+chromatography+coupled+with+ultra+violet+and+electrospray+ionization+ion+trap+detection+in+96-well+format.">Fast carbohydrate analysis via liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection in 96-well format.</a> J. Chromatogr. A. 1350, 44-50 (2014).</li></ol>

We have nothing to disclose.

Polysaccharide detection with the phenol-sulfuric-acid method: Different monosaccharides show different absorption maxima and molar extinction coefficients by use of this method9. This results in different absorption maxima of polymers, which contain several sugars in different amounts. The different wavelengths of absorption maxima for 16 different commercially available polymers are given in Table 5. The polymers were dissolved (1 g/L) in ddH2O, stirred (150 rpm) overnight and measured with the phenol-sulfuric-acid-method. Diutan gum showed the lowest absorption maxima at 470 nm and scleroglucan and hyaluronic acid the highest at 484 nm. Based on these results 480 nm was chosen for this screening platform. The relative absorbance of the polymers was calculated based on the absorbance obtained with 1 g/L glucose (set as 100%). The lowest results were obtained with scleroglucan and succinoglycan, both with 92%. This was expected because scleroglucan only contains glucose and succinoglycan contains glucose and galactose in a ratio of 7:1. Both commercial polymers have different losses of drying and different ash contents, this is the reason why the theoretical value of ~110% was not reached. Xylan showed the highest relative absorbance with 311%. The reason for this is the high molar extinction coefficient achieved from xylose due to the more dominant furanose form. At a level of 0.1 g/L glucose the quantification limit was reached, as well as the detection limit at a concentration &#60;0.05 g/L. However, the detection limit for positive strains in the screening is higher than 0.7 g/L and therefore, the assay showed a good performance. In order to get reliable results, the remaining glucose after gel-filtration was determined with a glucose-assay and this value was subtracted from the value from the phenol-sulfuric-acid method.
Automated glucose-assay dilution: The performance of the glucose determination after cultivation (dilution 1:100) was investigated. For this, 10 &#181;l of supernatant were transferred to 990 &#181;l of ddH2O in a deep well plate and mixed via ten times aspirating and dispensing 180 &#181;l out of this dilution. The second critical step was the correct pipetting of only 5 &#181;l aliquot for the 1:10 dilution from the glucose-assay after gel-filtration. In order to generate the dilution 25 &#181;l of ddH2O were transferred with a 50 &#181;l-tip first, afterwards 20 &#181;l of ddH2O and 5 &#181;l gel-filtrate were aspirated together. This ensures a better removal of the 5 &#181;l aliquot out of the tip. Both dilution steps were verified with various glucose standards via a glucose-assay. The results for two exemplary concentrations are given in Table 6. The 1:100 dilution for the determination of the glucose content after cultivation showed high precision for both standards with a CV &#60;1.2%. At the same time, the accuracy for the higher standard was up to 11.6 (% error). However, this is negligible as the glucose determination represents only the remaining glucose content after cultivation and therefore, is not important for the polymer detection. The 1:10 dilution for the remaining glucose after gel-filtration showed very reliable results with a CV &#60;1.4% and an accuracy &#60;2.1% error.
Consideration of evaporation: The screening requires 3.5 hr from the first step to the first glucose-assay. In order to find out, whether this time frame has an influence on uncapped MTP storage, 50 &#181;l of glucose-assay calibration standards were stored with and without cover for 3.5 hr in the robot carousel. In the calibration range (45 to 4.5 mg/L) the sample concentration hardly increased. An increase &#8212; caused by evaporation &#8212; was below 2.1% and only for the two lowest concentrations (1.8 and 0.9 mg/L) it reached up to 12.4% (Table 7).
Gel-filtration: High amounts of non-metabolized glucose disturb the quantitative detection of glucose from the hydrolyzed polymer. Therefore, a gel-filtration step was required to remove the remaining glucose after cultivation. Additionally, the gel-filtration purifies the polymer containing supernatant from salts and monomeric carbohydrate compounds, others than glucose, to minimize the analytical background in the monomer analysis. At the gel-filtration step 35 &#181;l of filtrate were placed in the center of the well. For validation of the robustness of gel-filtration in the automated system, eight calibration standards from 0.045 up to 9 g/L glucose were filtrated (n = 8). The glucose of every concentration was always reduced by more than 95% of the initial value (Table 8). In doing so, the gel-filtration showed very good results for various concentrations of glucose. Additionally, the remaining glucose after gel-filtration was also determined with a glucose-assay and subtracted from the phenol-sulfuric-acid determination to receive the correct amount of glucose equivalent for the hydrolyzed polymer.
Pyruvate-assay: First of all, it was investigated whether the neutralized and diluted (1:10) TFA-matrix from the hydrolysis step interferes with the enzymatic reaction. Therefore, the complete assay was performed twice, one time with and one time without matrix and showed reliable results. Finally, the pyruvate content of 16 commercially available polymers was successfully measured and is depicted in Figure 3. It is generally known that out of those 16 polymers only succinoglycan and xanthan naturally contain pyruvate. With our pyruvate-assay both of these polymers were correctly identified. In scleroglucan, welan gum and xylan pyruvate was also detected in significant amounts. Overall, the capability of the approach was validated and the pyruvate-assay showed a high performance. It proved to be able to detect pyruvate in different polymers after hydrolysis.
Carbohydrate fingerprint: After performing all analytical modules in the automated screening, potential EPS producers were selected for the carbohydrate fingerprint analysis. For this, several criteria were applied: 1) Positive observation of viscosity after centrifugation and/or after filtration. 2) Precipitation before and after filtration. Observed fibers and flakes were evaluated as positive. 3) Glucose equivalent value from the phenol-sulfuric-acid method. Values &#62;700 mg/L were rated as positive and values between 300 and 700 mg/L were rated as putative EPS producers. When two or three criteria were evaluated as positive, the strains were selected for further carbohydrate fingerprint analysis. The criteria can be customized towards the individual purpose of the EPS screening (e.g. low viscosity EPS). Our approach aimed at finding efficient EPS producers. When searching for strains that only produce small amounts of EPS the evaluation limit of the glucose equivalent should be reduced.
Technical benefit and future applications: One interesting feature of this protocol is the modular character of the steps and the different analytical modules. They can be combined in different ways, adjusted to individual requirements and novel modules can easily be implemented. Furthermore, the analytical modules can be used separately, e.g. the hydrolysis module in combination with the HT-PMP-derivatization module is able to perform a monomeric composition analysis from different polymer solutions (1 g/L) in 96-well format. For laboratories without having access to a liquid handling system the complete screening can be handled manually without any changes in the pipetting scheme. However, using a liquid handling system increases the throughput to up to 768 strains (instead of 192 strains if screened manually) per day. The protocol that is described here is capable of a screening for different genera and therefore, for the screening of large strain collections to identify novel EPS producers and analyze their carbohydrate fingerprint in one approach (Figure 4). Furthermore, a targeted screening for polysaccharides containing rare sugars like fucose, uronic acids or even unknown sugars can be performed via the detailed monosaccharide analysis. Also, different sugar combinations in defined ratios can be detected. This enables the simple identification of structurally related variants of already known EPS or novel EPS.

Microbial exopolysaccharides (EPS) are a structurally highly diverse group of polymers that fulfill various biological functions. They usually are built of complex repeat units, which are distinguished by different types of monomers (sugar, sugar derivatives, sugar acids), the bonds between these monomers and their substitutions. The structural diversity of microbial polysaccharides confers rather different characteristics to the members of this molecule class, which allows their application in different fields like food1, cosmetics2,3, construction chemistry4 or water treatment5. To further extend the field of application of these bio-based and such sustainable polymers the identification of novel natural microbial polysaccharides as well as the engineering of structural variants represents promising approaches. Either way, a fast screening method is required to quickly scan a vast number of microbial strains for their polysaccharide formation, and to analyze their products. Therefore, we have recently developed a 96-well HT-screening platform for the analysis of microbial polysaccharide production from natural isolates or engineered strain variants that includes the identification of the monomeric composition6.
Applying this platform for a first screening round of ~500 natural isolates allowed us to identify only about 10 to 20% of the isolated strains as being able to produce EPS (data not shown). This means that 80-90% of the analyzed strains did not produce EPS under the conditions applied, and therefore, a further analysis of the detailed carbohydrate fingerprint was not necessary. As this highly sophisticated identification of the monomeric composition is a time consuming process, especially for data analysis, a fast pre-screening method to identify the strains positive in EPS production, would drastically increase the efficiency. Furthermore, reagents, consumables and measurement time at the UHPLC-UV-ESI-MS would be reduced. Additionally, the different analytical modules, while on one hand make the method highly reliable, are on the other hand complicating the manual handling of more than two 96-well plates in parallel, and as such restrict the full potential of the method. For these reasons, we decided to develop an automated screening platform. Therefore, we combined the modular format of the existing carbohydrate fingerprint technique with a fully automated fast detection method of the total sugar content, based on absorbance measurement.
The phenol-sulfuric-acid method still represents the method of choice for the fast determination of total carbohydrate content of bacterial and plant polysaccharides7,8. This method was first described by Dubois et al.9 and adapted for different applications and sample sizes, even for small scale in 96-well plates10,11. The phenol-sulfuric-acid method measures the total carbohydrate content by one value, summating all monomeric, oligomeric and polymeric carbohydrates of the samples.
Taking these aspects into account, the choice of a suitable cultivation medium is essential to apply this method. Complex media containing oligomeric or polymeric carbohydrate compounds like yeast extract might lead to an altered polymer content and therefore, should strictly be avoided. Furthermore, high amounts of sugars are used as C-source for the cultivation of the strains. High levels of remaining carbohydrates from the cultivation process might negatively interfere with the measurement of the EPS content.
Therefore, the use of defined and pure sugars is advised. In our experiments we used glucose for the cultivation of the cells. The remaining glucose after cultivation was reduced via a gel-filtration and determined via a glucose-assay. Finally, the glucose equivalents of the polysaccharides were calculated by subtracting the remaining glucose after the gel-filtration from the total carbohydrate content that had been detected with the phenol-sulfuric-acid method. Gel-filtration and glucose-assay in combination with the phenol-sulfuric-acid method ensure reliable results and are capable of being our first, completely automated detection system.
Two new analytical modules were included in the automated screening platform to increase the amount of information from the EPS-detection system: the precipitation and the observation of an increase in viscosity.
Many different EPS &#8212; e.g. succinoglycan, xanthan and colonic acid12&#160;&#8212; are reported to be modified with a non-carbohydrate pyruvate ketal on sugar positions C4 and C6. Those pyruvate ketals (just as succinyl half esters and uronic acids) contribute to the polyanionic nature and therefore, to the physical properties of the polymer by interacting via divalent cation bridges13. In order to identify those particular polymers the determination of pyruvate was established as another additional analytical module. This increases the information about the polysaccharide substituents and their potential macroscopic properties.
Combining all the modules enables the identification of different EPS as well as a fast and efficient determination of EPS producers. By that approach the screening platform could be divided into two major parts (Figure 1). Within the automated screening (part I) the workflow occurs fully automated (Table 1) to quickly preselect the EPS producing strains. The carbohydrate fingerprint analysis (part II) quantitatively determines the monomer composition of the EPS produced by the preselected strains. Thereby, the data analysis was minimized in order to optimize the screening of large strain collections. This offers the possibility to analyze 384 strains in one single automated screening run and with two runs that are possible per day of 768 strains per day. Additionally, the carbohydrate fingerprint analysis gives an even more detailed overview of all the identified EPS. This enables the directed analysis and identification of only slightly differing EPS variants or completely new ones compared to the already described chemical structures of EPS.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="126">
			<td height="126" style="height:126px;width:335px;">96 well deep well plate 2.0 mL (DWP)</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">780271</td>
			<td style="width:224px;">Main-culture (CP 1-1 to 1-4), equilibration plates for gel-filtration (CP 1-6, 1-7, 2-6 and 2-7), 1:100 dilution for the&#160; glucose assay (CP 4-1 to 4-4) containing 990&#181;l of ddH2O</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Breathable Sealing Film</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">BF-400-S</td>
			<td style="width:224px;">Incubation film for pre- and main-culture DWP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Aluminum Sealing Film</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">PCR-AS-200</td>
			<td style="width:224px;">-80 &#176;C storage film for glycerol-stock plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">MCA96 Nested Disposable Tips 200 &#181;l</td>
			<td style="width:185px;">TECAN</td>
			<td style="width:143px;">30038619</td>
			<td style="width:224px;">Worktable position (WTP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">A/B glass-filter-plate 1&#181;m</td>
			<td style="width:185px;">Pall Corporation</td>
			<td style="width:143px;">PN 8031</td>
			<td style="width:224px;">Stored on collector plate (CP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well micro titer plate V-Bottom</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">651201</td>
			<td style="width:224px;">Collector plate for filtration plate (CP 2-1 to 2-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well SpinColumn G-25</td>
			<td style="width:185px;">Harvard Apparatus</td>
			<td style="width:143px;">74-5612</td>
			<td style="width:224px;">Stored on washing DWP (CP 1-6, 1-7, 2-6 and 2-7)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well micro titer plate V-Bottom</td>
			<td style="width:185px;">Nunc</td>
			<td style="width:143px;">249944</td>
			<td style="width:224px;">Collector plate for gel-filtration plate (CP 3-1 to 3-4)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Nested Disposable Tips SBS 50 &#181;l tips</td>
			<td style="width:185px;">TECAN</td>
			<td style="width:143px;">30038609</td>
			<td style="width:224px;">Carousel position (CP 4-6, 4-7 and 5-1 to 5-6)</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:335px;">Trough 250 ml</td>
			<td style="width:185px;">Axygen</td>
			<td style="width:143px;">Res-SW96-HP</td>
			<td style="width:224px;">Water WTP 1-1, Glucose assay reagent-mix WPT 1-2, ammonium-acetat buffer pH 5.6 (CP 5-7)</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:335px;">96-well micro titer plate F-Bottom (MTP)</td>
			<td style="width:185px;">Greiner Bio-One</td>
			<td style="width:143px;">655101</td>
			<td style="width:224px;">precipitation 1 (CP 6-1 to 6-4), pH-value (CP 7-1 to 7-4), precipitation 2 (CP 8-1 to 8-4), phenol-sulfuric-acid method (CP 8-5 to 8-8), glucose-assay (CP 9-1 to 9-9), dummy plates (WTP 3-1, 3-2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">96-well silicon cap mat&#160;</td>
			<td style="width:185px;">Whatmann</td>
			<td style="width:143px;">7704-0105</td>
			<td style="width:224px;">Cover mat for MTP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">200 &#181;l pipette tips</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:143px;">70.760.002</td>
			<td style="width:224px;">For manually handling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">1000 &#181;l pipette tips</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:143px;">70.762</td>
			<td style="width:224px;">For manually handling</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">96-well-PCR micro titer plate&#160;</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">781350</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatisation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">TPE (thermoplastic elastomer) cap mat&#160;</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">781405</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatisation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:335px;">Filter plate 0.2&#160;&#181;m Supor,&#160;</td>
			<td style="width:185px;">Pall Corporation</td>
			<td style="width:143px;">PN 8019</td>
			<td style="width:224px;">Filtration of samples for UHPLC-ESI-MS analysis with a MTP collector plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Pipette Tips LHS 5-300 &#181;l</td>
			<td style="width:185px;">Brand</td>
			<td style="width:143px;">732150</td>
			<td style="width:224px;">Brand LHS system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">ABTS (2.2-azino&#8209;bis-(3&#8209;ethylbenzthiazoline)-6-sulfonic&#160;acid)</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">A1888</td>
			<td style="width:224px;">Glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Glucose oxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">G2133</td>
			<td style="width:224px;">Glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Horseradish peroxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6782</td>
			<td style="width:224px;">Glucose-assay, pyruvat-assay</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:335px;">DA-64 (N-(Carboxymethylaminocarbonyl)-4.4&#39;-bis(dimethylamino)-diphenylamine sodium salt)</td>
			<td style="width:185px;">Wako Chemicals GmbH</td>
			<td style="width:143px;">043-22351</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Pyruvate oxidase&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4591</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Potassium phosphate dibasic</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">P749.3</td>
			<td style="width:224px;">Pyruvat- and glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Potassium phosphate monobasic</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">3904.3</td>
			<td style="width:224px;">Pyruvat- and glucose-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Thiamine pyrophosphate</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">C8754</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Magnesium chloride hexahydrate</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">31413</td>
			<td style="width:224px;">Pyruvat-assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">2-Propanol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20922.466</td>
			<td style="width:224px;">Precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Phenol</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20599.231</td>
			<td style="width:224px;">Phenol-sulfuric-acid method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Sulfuric acid</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">4623.4</td>
			<td style="width:224px;">Phenol-sulfuric-acid method</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Trifluoroacetic acid</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">T6508</td>
			<td style="width:224px;">Hydrolysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Ammonium solution</td>
			<td style="width:185px;">Carl-Roth</td>
			<td style="width:143px;">P093.1</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Ethanol absolut</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20821.321</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:335px;">Phenol red&#160;</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:143px;">B21710</td>
			<td style="width:224px;">Hydrolysis, PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">1-Phenyl-3-methyl-5-pyrazolone</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">M70800</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Methanol LC-MS</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">83638.320</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Acetonitril LC-MS</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">83040.320</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Acetic acid</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:143px;">338826</td>
			<td style="width:224px;">PMP-derivatization,</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Ethanol absolut</td>
			<td style="width:185px;">VWR</td>
			<td style="width:143px;">20821.321</td>
			<td style="width:224px;">PMP-derivatization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Methyl red&#160;</td>
			<td style="width:185px;">Alfa Aesar</td>
			<td style="width:143px;">36667</td>
			<td style="width:224px;">pH-value</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Robotic liquid handling system&#160;</td>
			<td>Tecan</td>
			<td>Freedom EVO</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid handling station LHS</td>
			<td>Brand</td>
			<td>709400</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tip-Adapter&#160;</td>
			<td>Brand</td>
			<td>709434</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Ends MC 20-300&#181;L</td>
			<td>Brand</td>
			<td>709416</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adapter 60mm</td>
			<td>Brand</td>
			<td>709430</td>
			<td style="width:224px;">Worktable setup in Figure 2</td>
		</tr>
	</tbody>
</table>

automated modular high throughput exopolysaccharide screening platform coupled with highly sensitive carbohydrate fingerprint analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

Watch this Scientific Journal Video about Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis at JoVE.com

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

automated-modular-high-throughput-exopolysaccharide-screening

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Biochemistry of the Cell

SCIENTISTS IN ACTION

11.4K Views.  Technische Universität München. We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Video: Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...