이 작품은 고든 무어와 베티 재단에 의해 및 UNCF / 머크 과학 이니셔티브에 의해 투자되었다.

1. 심사 플레이트 준비 <ol><li> 25g LB 성장 매체, 15g 한천과 증류수 1L에 1.5g 카르복시 메틸 셀룰로오스 (CMC) 기판 및 소독에 압력솥을 추가합니다. </li><li> autoclaving 후 적절한 항생제를 냉각하고 추가할 수 있도록 중간, 그리고 100μM의 최종 농도 IPTG. </li><li> 각 5 큰 사각형 배양 접시 / bioassay 트레이 (240 X 240 X 20mm 또는 더 큰)에 200mL 매체를 하거라. 접시가 완전히 건조하도록 허용합니다. </li></ol> 2. Endoglucanase 라이브러리 생성 및 화면 <ol><li> endoglucanase 촉매 도메인 (들)의 라이브러리를 생성합니다. 이것은 여러 가지 방법으로 수행할 수 있습니다 (예 : 사이트 이동 또는 임의의 돌연변이 유발, 재조합 등)는 연구자에 의해 결정됩니다. </li><li> 그들은 락 연산자와 T7 프로 모터의 제어하에있는 것으로 같은 벡터로 라이브러리를 복제하고, periplasm 타겟팅을위한 pelB 신호 시퀀스가​​ 포함되어 있습니다. 적당한 벡터의 예입니다 Novagen에서 pET22b (+)입니다. </li><li> E.에 endoglucanase 라이브러리를 변환 대장균은 BL21 (DE3)과 하나의 식민지에 대한 판 변형. </li><li> 37 박 변환을 품어 ° C. </li><li> 무균 이쑤시개를 사용하여 적절한 항생제로 보충 400μL LB 매체를 포함하는 96 잘 깊은 잘 접시에 클론의 예방. 37 품어 ° C 250 rpm으로 잡고 하룻밤. </li><li> -80에서 장기 저장을 위해 10 %의 최종 농도 96 ° C.에 잘 하룻밤 문화 살균 글리세롤 추가 이것은 마스터 판 것입니다. </li><li> 96 핀 스탬프를 사용하여, IPTG와 CMC를 포함한 심사 접시에 접시 야간 문화를 복제합니다. 최대 숙박 문화의 5 세트는 각 심사 플레이트에 찍혀 있습니다. </li><li> 각 96 잘 접시의 정체성과 방향이 알려져 있도록 심사 번호판을 레이블. </li><li> 실내 온도 (17-25 ° C) 숙박 또는 볼 식민지가 형성 때까지에서 스탬프 라이브러리를 품어. </li><li> 식민지의 모든 흔적을 제거 때까지 증류수로 접시를 씻어. </li><li> 세탁 판에; 붓고 콩고 레드 (물 속에 0.5 % CR). 접시를 충당하기 위해 충분 CR 추가되었는지 확인하십시오. </li><li> 실온에서 15 분 알을 품다. CR 보육을위한 15 분 초과하지 마십시오. 긴 배양 시간이 덜 뚜렷한 halos집니다. </li><li> CR을 붓고 및 1M NaCl의 풍부한 양의 (이상 충분히 번호판을 충당하기 위해) 추가합니다. </li><li> 실온에서 15 분 알을 품다. </li><li> CMC 열화 halos을 나타내기 위해 NaCl을 하거라. halos보다 해상도 들어, NaCl 또는 여러 NaCl 세척과 함께 더 이상 부화 시간이 필요할 수 있습니다. </li><li> 멸균 이쑤시개를 사용하여 더욱 특성화를위한 마스터 플레이트에서 활성 효소의 클론을 선택하십시오. </li></ol> 3. 대표 결과 : 이 높은 처리량 화면 판독의 예제는 그림 3에 표시됩니다. 클론은 저하 영역의 크기에 따라 비활성, 약하게 활성, 그리고 높은 활성 상태로 확인할 수 있습니다. 알려진 활동의 효소가 참고로 라이브러리에 포함되어있는 경우 특히 유용합니다. 여기에 그림과 같이, 활성 효소의 식별은 강력하고 재현할 수 있습니다. 그러나, 심사 플레이트의 적절한 라벨이 매우 중요하다는 것을 양해해 주시기 바랍니다. 연구원 ° C 자세한 특성화를위한 -80에서 저장된 마스터 플레이트에서를 선택하기 위해, 멀리 식민지를 세척 후 Active 변종을 식별할 수 있어야합니다. 마지막으로, 그것은 선험에게 인공 대 자연의 기판에 활동 사이의 관계를 평가하기 어렵습니다. 따라서 연구자들은 단백질 적합성을 결정하는 산업상 관련 자료에있는 모든 긍정적인 클론을 테스트해야합니다. <img src="/files/ftp_upload/2942/2942fig1.jpg" alt="그림 1"/> 그림 1. 심사 절차의 ​​도식 개요. 세포 endoglucanase 라이브러리로 변환 및 단일 식민지에 대한 선택 도금입니다. 클론 -80 (1) 저장 ° C와 endoglucanase 표현을 유도하기 위해 CMC와 IPTG를 포함하는 접시에 (2) 복제 도금에 대한 96 자 딥 웰 플레이트에 야간 들어서 성장. 활성 클론은 -80 ° C.에 저장된 마스터 플레이트에서 선택되고 <img src="/files/ftp_upload/2942/2942fig2.jpg" alt="그림 2"/> 그림 2. E.에 곰팡이 촉매 도메인의 복제와 표현 대장균. (A) Endoglucanase 표현 벡터가. 유전자는 T7 프로 모터의 제어하에 pET22b (+) (Novagen)에 복제되었습니다. (B) 오른쪽 패널 : endoglucanase - 표현 BL21 (DE3) 식민지. 왼쪽 패널 : 콩고 레드와 세탁 및 개발 후 CMC 열화 halos. <img src="/files/ftp_upload/2942/2942fig3.jpg" alt="그림 3"/> 그림 3. CMC / 콩고 레드 화면에서 샘플 결과입니다. 사진은 세탁과 콩고 레드 개발 후 심사 번호판을 찍어. S에있는 모든 식민지creening 번호판과 비슷한 크기의되었습니다.

<ol>
	<li>Atsumi, S., Hanai, T., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fermentative+pathways+for+synthesis+of+branched-chain+higher+alcohols+as+biofuels.">Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.</a> Nature. 451, 86-89 (2008).</li><li>Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulases+and+biofuels.">Cellulases and biofuels.</a> Curr Opin Biotechnol. 20, 295-299 (2009).</li><li>Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+endoglucanase+II+from+Trichoderma+reesei+to+improve+the+catalytic+efficiency+at+a+higher+pH+optimum.">Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.</a> J Biotechnol. 135, 190-195 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+endoglucanase+III+(Cel12A)+from+Trichoderma+reesei.">Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.</a> Appl Microbiol Biotechnol. 83, 649-657 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+thermostable+fungal+cellulases+created+by+structure-guided+recombination.">A family of thermostable fungal cellulases created by structure-guided recombination.</a> Proc Natl Acad Sci. 106, 5610-5615 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCHEMA+recombination+of+a+fungal+cellulase+uncovers+a+single+mutation+that+contributes+markedly+to+stability.">SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.</a> J Biol Chem. 284, 26229-26233 (2009).</li><li>Lantz, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrea+jecorina+CEL6A+protein+engineering.">Hypocrea jecorina CEL6A protein engineering.</a> Biotechnol Biofuels. 8, 3-20 (2010).</li><li>Mahadevan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+and+CBM+engineering+of+Cel5A+(Thermotoga+maritima).">Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).</a> FEMS Microbiol Lett. 287, 205-211 (2008).</li><li>Nakazawa, H. <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+the+catalytic+domains+of+Trichoderma+reesei+endoglucanase+I,+II,+and+III,+expressed+in+Escherichia+coli.">Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli.</a> Appl Microbiol Biotechnol. 81, 681-689 (2008).</li><li>Wang, H., Jones, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Macrophomina+phaseolina+endoglucanase+(EGL+1)+gene+product+in+bacterial+and+yeast+expression+systems.">Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.</a> Appl Biochem Biotechnol. 81, 153-160 (1999).</li><li>Howard, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+activity+of+a+Phanerochaete+chrysosporium+cellobiohydrolase+(CBHI.1)+expressed+as+a+heterologous+protein+from+Escherichia+coli.">Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli.</a> Afr J Biotechnol. 2, 296-300 (2003).</li><li>Howard, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a+chimeric+Phanerochaete+chrysosporium+cellobiohydrolase+expressed+from+Escherichia+coli.">Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli.</a> Afr J Biotechnol. 3, 349-352 (2004).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Appl Env Microbiol. 43, 777-780 (1982).</li><li>Gilkes, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+and+substrate+specificities+of+cellulases+from+cloned+bacterial+genes.">Mode of action and substrate specificities of cellulases from cloned bacterial genes.</a> J Bio Chem. 259, 10455-10459 (1984).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기에서 설명한 프로토콜은 최소한의 조작으로 활성 효소의 신속하고 높은 처리량 신분증을 수 있습니다. 활동 검출은 매우 민감하고, 질적으로 세포 내에서 활동의 양을 나타냅니다. 사용의 용이성은 E.의 기능적 표현에 의해 제한 효소 라이브러리의 광범위한이 방법은 적합 대장균. 또한 이런 종류의 활동 심사는 곰팡이 cellulases에 국한되지 않고 있지만 endoglucanase의 활동에 적용?...

<table border="1"><tbody><tr><th> 시약 </th><th> 회사 </th><th> 제품 번호 </th></tr><tr><td> IPTG </td><td> 시그마 </td><td> I1284 </td></tr><tr><td> 콩고 레드 </td><td> 시그마 </td><td> C6277 </td></tr><tr><td> 카르복시 메틸 셀룰로오스 </td><td> 시그마 </td><td> 360384 </td></tr><tr><td> NaCl </td><td> 시그마 </td><td> S1679 </td></tr><tr><td> Bacto - 한천 </td><td> BD Biosciences </td><td> 214030 </td></tr><tr><td> Bioassay 플레이트 </td><td> 써모 과학 </td><td> 240845 </td></tr></tbody></table>

high throughput screening of fungal endoglucanase activity in escherichia coli

Cellulase 효소 (endoglucanases, cellobiohydrolases 및 β - glucosidases) 차례로 연료 알콜 1로 변환할 수 있습니다 구성 요소 당분로 hydrolyze의 셀룰로오스. 신재생 에너지를 제공하기 위해 cellulosic 바이오 매스의 효소 가수 분해에 대한 잠재력은 경제적인 연료 생산 2 cellulases을 엔지니어 노력을 강화하고있다. 특히 관심이 이미 식품, 섬유 처리에 대한 산업상 사용되는 곰팡이 cellulases 3-8은 다음과 같습니다. 돌연변이 cellulases의 라이브러리 간의 적극적인 변종을 식별하는 것은 기술 프로세스에 중요합니다, 적극적인 돌연변이가 더욱 향상된 특성 및 / 또는 추가 테스트 돌연변이 유발을 받게 될 수 있습니다. 곰팡이 cellulases의 효율적인 엔지니어링은 기본 생물에 대한 유전자 도구의 부족으로하고 heterologous 호스트에있는 효소를 표현의 어려움에 의해 방해되었다. 최근 모리카와 동료 E.의 표현 방법을 개발 대장균 H. jecorina 3,9, 대량으로 cellulases을 분비하는 능력과 함께 중요한 산업 곰팡이에서 endoglucanases의 촉매 도메인. 기능성 E. 대장균 표현도 Macrophomina phaseolina 10 Phanerochaete chrysosporium 11-12 포함한 다른 곰팡이에서 cellulases에 대한보고되었습니다. 우리는 E.에 곰팡이 endoglucanase 활동의 높은 처리량 검사에 대한 방법을 제시 대장균. (그림 1)이 방법은 고체 매체에 성장 세포에 의해 카르복시 메틸 셀룰로오스 (CMC)의 효소 저하를 시각화하는 일반적인 미생물의 염색 콩고 레드 (CR)를 사용합니다. 활동 분석은 저렴한 시약, 최소한의 조작을 필요로하고, 식민지 사이트에서 열화의 지역 ( &quot;halos&quot;)로 모호하지 않은 결과를 제공합니다. 효소 활동의 양적 측정이 방법에 의해 결정되지 않을 수 있지만, 우리는 헤일로 크기가 셀에 총 효소 활동을 상호 것으로 나타났습니다. 개인 긍정적인 클론의 추가 특성은 상대적으로 단백질 적합성을 결정합니다. 전통적인 박테리아 전체 셀 CMC / CR 활동 assays 13 교차 오염, 또는 대규모 실험을 덜받을 수있는 것입니다 CMC 한천 우물에서 잠복기 문화의 적용을받습니다 식민지, 한천에 포함된 CMC를 따르고 포함됩니다. 여기 cellulase 활동 14 기존의 세척 방법을 수정 향상된 프로토콜을보고 : CMC 한천 플레이트에 성장 세포는 이전에 CR의 얼룩을 제거됩니다. 우리 프로토콜은 크게 교차 오염을 줄이고 클론 수천의 급속한 심사를 허용, 확장성이 높은 것입니다. H. 이외에 jecorina 효소, 우리가 표현하고이 프로토콜은 생물의 범위에서 효소에 적용할 수 있다고 제안, Thermoascus aurantiacus 및 Penicillium decumbens (그림 2 참조)에서 endoglucanase 변종을 검사.

Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

우리의 곰팡이 endoglucanase 활동을위한 화면으로 저렴한 비용, 높은 처리량 방법을 설명<em&gt; E. 대장균.</em&gt; 방법은, 기판 저하의 단순한 시각적 판독에 의존 효소 정화를 필요로하지 않으며, 확장성이 높은 것입니다. 이것은 효소 변종 대형 도서관의 신속한 심사를 위해 수 있습니다.

에서 곰팡이 Endoglucanase 활동의 높은 처리량 검사 대장균</em

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

high-throughput-screening-fungal-endoglucanase-activity-escherichia

Research

JoVE Journal

Biology

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

20.5K 조회수.  California Institute of Technology. 우리의 곰팡이 endoglucanase 활동을위한 화면으로 저렴한 비용, 높은 처리량 방법을 설명 E. 대장균. 방법은, 기판 저하의 단순한 시각적 판독에 의존 효소 정화를 필요로하지 않으며, 확장성이 높은 것입니다. 이것은 효소 변종 대형 도서관의 신속한 심사를 위해 수 있습니다.

비디오: High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

의 활동을 측정하는 어세이 대장균은 라이신 Decarboxyase을 Inducible

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

연구

생물학

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.

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스

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

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

안쪽 정류기의 칼륨 채널의 소형 분자 변조기에 대한 높은 처리량 검사

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

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

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electrocompetent의 준비를위한 신속한 프로토콜<em&gt; 대장균</em&gt;와<em&gt; 콜레라 균</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

라이브의 검출<em&gt; 대장균</em&gt; O157 : PMA-qPCR에 의한 H7 셀

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

에서 생산 재조합 이황화 풍부한 독 단백질에 적용되는 높은 처리량 정량 식 검사 및 정화<em&gt; E. 대장균</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

단백질 공동 식의 다각적 인 장점에<em&gt; 대장균</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

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.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

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

에서 곰팡이 Endoglucanase 활동의 높은 처리량 검사 대장균

요약

더 많은 비디오 탐색

이 비디오의 챕터

의 활동을 측정하는 어세이 대장균은 라이신 Decarboxyase을 Inducible

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스

안쪽 정류기의 칼륨 채널의 소형 분자 변조기에 대한 높은 처리량 검사

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

Electrocompetent의 준비를위한 신속한 프로토콜

라이브의 검출

에서 생산 재조합 이황화 풍부한 독 단백질에 적용되는 높은 처리량 정량 식 검사 및 정화

막 단백질의 녹색 형광 단백질 기반의 표현 상영에

단백질 공동 식의 다각적 인 장점에

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사