이 작품은 에너지의 미국학과, 기본 에너지 과학 부문의 사무실에서 미국 에너지 대호 Bioenergy 연구 센터 계열 (과학 BER의 DOE 사무소 DE - FC02 - 07ER64494)과 부여 DE - FG02 - 91ER200021에 의해 부분적으로 투자되었다 화학 과학, Geosciences 및 Biosciences.의 우리는 그들의 자료와 개념적 공헌에 대한 존 스콧 - 크렉과 멜리사 Borrusch 감사드립니다.

1. 효소 생산 <ol><li> 벡터 pPICZ 또는 pPICZα에 해당 유전자의 복제와 P.으로 변환하여 Pichia pastoris에의 단백질을 생산 pastoris. 메탄올 유도와 함께 실패하는 500 ML flasks 300 - ML 배치에서 세포를 성장마다 24 시간 (Banerjee 외., 2010a). </li><li> 집중과 접선 흐름 여과하여 문화 filtrates을 탈염하다. </li><li> -80에서 20 % 글리세롤의 작은 aliquots에있는 효소를 저장 ° C. 농도 범위는 MG / ML 10-10입니다. </li></ol> 2. 실험의 설계 <ol><li> 입력 테스트하는 효소의 수를 그들의 위턱과 아래턱 비율 (핵심 효소 5 %의 낮은 비율을 예), 그리고 디자인 Expertsoftware으로 실험 설계 (예 : 차 또는 입방)의 유형입니다. </li><li> 15 MG / G 글루칸 총 로딩을 추가할 수 각 효소 μg에 금액을 계산하고 다음에 추가 μl 각 효소의 양을 계산각각의 반응. </li><li> 실험 설계 및 Biomek FXP 소프트웨어로 가져오기를 전송 -에서 - 파일 함수를 사용에 따라 물, 효소의 분배에 대한 소스 labware, 소스 우물, 대상 labware, 대상 우물, 그리고 전송 볼륨을 지정 Excel 워크시트를 생성합니다. </li></ol> 3. 효소 가수 분해 <ol><li> 패들 저수지에서 5 μg / ML의 테트라 사이클린과 5 μg / ML cycloheximide를 포함, 50 MM 나트륨 구연산, 산도 4.8의 바이오 매스 슬러리를 일시 중지합니다. </li><li> 96 - 웰 깊은 잘 반응 플레이트의 각 우물에 슬러리의 Robotically pipet 200 μl는 스팬 - 8로 잘라 마지막 0.5 cm로 1000 μl pipet 도움말을 사용합니다. 100 μl / 초 한 번에 슬러리를 혼합하고 패들 저수지에서 동일한 볼륨을 기음하고 접시에 분배. </li><li> 베크 만 FX에 입력된 프로그램을 사용하여 동일한 접시에 효소를 분배. </li><li> pierceable capmats로 번호판을 밀봉합니다. </li><li> 반전과 탭접시는 몇 시간은 표면 장력을 극복하기. 10 RPM의 회전 하이브 리다이 제이션 인큐베이터에서 48 시간 50 ° C에서 접시를 놓습니다. </li></ol> 4. Glc 및 Xyl의 결정 <ol><li> 스윙 버킷 원심 분리기의 1,250 XG에서 3 분 반응 번호판을 원심 분리기. </li><li> Biomek FX의 AP96 포드를 사용하여 정기적으로 96 - 웰 플레이트로 뜨는 100 μl을 전송합니다. </li><li> 90-95시 번호판을 품어 ° C 효소를 inactivate 후 30 초에 대한 1,250 XG에서 원심을 위해 10 분. </li><li> Glc 측정, 일반 96 - 웰 플레이트에 supernatants 12 μl를 전송합니다. 포도당 산화 효소 / 퍼옥시데이즈 (GOPOD) 시약의 192 μl를 추가합니다. 20 분 50 ° C에서 접시를 부화하고 microplate 리더에서 510 nm의에서 absorbances를 참조하십시오. 공백없이 효소와 반응 혼합물이다. </li><li> Xyl 측정, 384 잘 접시에 4 μl를 전송합니다. 분석 시약을 추가하고 340 nm의에서 접시를 참조하십시오. 공백없이 효소와 반응 혼합물이다. </li></ol> 5. 데이터 분석 <ol><li> 알려진 Glc하고 원래 바이오 매스의 Xyl 내용에 따라 자신의 % Glc 및 Xyl의 수율로 흡광도 값을 변환합니다. 답변으로 디자인 전문 소프트웨어로 이러한 비율을 가져옵니다. 강력한 모델에 대한 기준이 충족되어 있는지 확인하는 통계 paramenters를 확인합니다. </li><li> 실험적으로 최고의 모델 예측을 확인합니다. </li></ol> 6. GENPLAT와 대표 검색 결과 : (1) 개인 순수한 단백질의 최적화된 혼합물의 창조. 결과는 효소가 중요있는 표시, 그리고 어떤 비율로, 또한 어떤 효소는 중요하지 않습니다. T. 풍부 일부 단백질 이러한 Cip1 및 Cip2로 reesei secretome은 (Nagendran 외., 2009), 암모니아 섬유 확장 (AFEX) 또는 알칼리 hyd에 의해 pretreated 옥수수 여물에서 Glc 릴리스에 역할을하지 나타납니다<html> 로겐, 과산화수소 (AHP) (Banerjee 외., 2010b). 반면에, 일부 단백질이 예기치 않게 중요합니다. 예를 들어, Glycosyl 하이드 롤 레이스 가족 10 11 엔도 - xylanases는 Glc 릴리스 모두 중요하다, 한 xylanase이 다른 (. Banerjee 외, 2010b, C)를 대신할 수 없습니다. Cel61A, T.의 사소한 단백질 reesei secretome 아주 Glc 중요하지만 릴리스 Xyl되지 않습니다. 다른 하나 또는 다른 (Banerjee 외., 2010c)에 필요한 반면 일부 효소는 Glc 및 Xyl 모두 릴리스 중요합니다. 0.94 MG / ML (즉, 비 최적화된 혼합물)의 농도 16 구성 요소 각각을 포함하는 합성 혼합은 AFEX - pretreated DDG에서 사용할 수있는 Glc의 38 %를 발표했다. 동일한 가수 분해 조건 하에서 최적의 혼합이되는 16 구성 요소의 농도가 0퍼센트에서 32 %로였다 (그림 2) Glc의 52 %를 발표했다. 이 실험은 정의 혼합 건설의 유틸리티를 보여줍니다. _content &quot;다른 전처리 / 기판 조합에 최적화된 칵테일&gt; (2) 창조. 현재 상업 cellulase의 준비은&quot;한 - 크기 - 맞는 - 모든 &quot;. 현실에서 최고의 칵테일은 기판과 전처리 모두에 따라 달라집니다. 하나의 제품 Accellerase 1000 같은 방법으로 pretreated 다양한 기판에서 서로 다른 pretreatments (그림 1)에 노출 동일한 기판 다른 수율을 제공 등. 우리는 여러 pretreatments 및 mutiple feedstocks (Banerjee 동부 표준시에 대한 순수한 효소의 혼합물을 최적화하는 GENPLAT를 사용했습니다 알., 2010c). 그림 2는 16 순수한 효소의 최적 효소 비율이 AFEX - pretreated 옥수수 여물, AHP - pretreated 옥수수 여물, 그리고 AFEX - pretreated DDG에 대한 다른 방법을 보여줍니다. 단 11 효소가 그림에 표시됩니다. 2 최적의 이유 나머지 다섯 (Cel61B, AbfB, Cip1, Cip2, 그리고 Cel12A)의 비율은 (Banerjee 외., 2010c) 0 %가된다는 것을 발견했다. (3) 소설 효소 발견 </str긴 사연이&gt;. 순수한 단백질의 최적화된 혼합물의 건설 진행 과정입니다. 새로운 단백질은 순수 양식 (상용 소스에서 heterologous 표현에 의해, 또는 정화하여)에서 사용할 수 해짐에 따라, 그들은 체계적으로 현재 최적화된 설정을 추가하여 테스트할 수 있습니다. Glc 또는 Xyl에 기여하는 새로운 단백질이 새, 최적화된 집합의 일부가 공개. 이러한 방법으로 GENPLAT는 바이오 매스 해체에 대한 새로운 효소의 식별을위한 플랫폼이된다. 우리는 T. 이외 버섯의 추출물을 만들었습니다 reesei와 A. 니제르는 다른 기판에 성장. 핵심 세트에 추가하면 하나의 특정 추출물 Glc 수율이 없어졌다. 책임 단백질은 정제과 (Banerjee와 월튼, 발표되지 않은 결과) 상업적 효소 준비에 존재하지 않는 소설 glycosyl 하이드 롤 레이스로 표시되었다. 더 효소 (4) 발견. 의 저하에 대한 효소가 가장 중요 GENPLAT로 표시하는 데특히 바이오 매스, 우리는 효소가 개선 초점되어야하는 더 나은 이해를했습니다. 중요한 효소의 더 나은 예는 자연에서 검색하여 발견되거나 단백질 공학에 의해 만들 수 있습니다. (효소가 원하는 속성에 따라 여러 가지 방법으로 자사의 T. reesei의 상동체보다 &quot;더&quot;가 될 수있다, 예를 들어, 높은 특정 활동, 큰 시너지 효과는 proteolytic 감도, 또는 향상된 열적 안정성을 감소). 순수한 셀룰로스 또는 기타 인공 기판과 결과는 자연 리그노셀룰로오스성에 관련되지 않을 수 있기 때문에 그것이 현실 복잡한 칵테일 (아무 바이오 매스 효소가 격리 작동하지 않기 때문에 중요한) 중요한 현실적인 기판 (활용하기 때문에 GENPLAT는 잠재적으로 더 효소를 시금위한 의미있는 플랫폼을 제공합니다 자료). (5) 상업 효소 최적화. 순수한 효소의 충분히 많은 양의 아직 현실 세계에서 존재하지 않습니다. 그들이 때까지 에탄올 프로ducers는 Accellerase 1000 MultifectXylanase와 같은 상용 제품에 의존해야합니다. 단, 상업 효소의 혼합물은 일반적으로 개인보다 더 나은 수행하고, GENPLAT은 여러 상업 효소의 최적 칵테일을 디자인하는 데 사용할 수 있습니다. 그림 3 네 상업 효소 준비의 혼합물을 최적화할 수 GENPLAT의 사용을 보여줍니다. AFEX - pretreated DDG에서 Glc의 릴리스에 대한 최적의 비율은 47% Accellerase1000, 27% Multifect Pectinase, 22% Novozyme 188, 그리고 4퍼센트 Multifect Xylanase (Banerjee 외., 2010c)으로 발견되었다. 그림 3B 네 효소 준비와 AFEX - DDG에서 Glc 수율의 차이를 보여줍니다, 최적화된 상업 혼합물은 높은 Glc 수율을 제공 혼자 Accellerase1000보다 SpezymeCP 혼자, 또는 16 요소 합성 혼합물. 이 실험은 또한 16 구성 요소 합성 혼합물은 상업 효소 중 하나 이상에 존재하는 최적의 Glc 릴리스에 필요한 하나 이상의 효소가없는 것을 나타냅니다. GENPLAT 우리 수에드는 구성 요소를 식별합니다. <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="그림 1"/> 그림 1. 하나가 상업 효소 준비는 모든 기판 모든 pretreatments 최적 않습니다. 다섯 기판 세 pretreatments은 (0.5 mm 입자 크기) 연삭 비슷한 조건 하에서 비교했다, 효소로드 (15 MG / G 글루칸) 및 가수 분해 조건 (48 시간, 50 ° C) 다른 AFEX - pretreated 기판. A.의 소화 Accellerase 1000 &#39;와. 옥수수 여물의 B.는 소화 Accellerase 1000&#39;와 세 가지 방법 (Banerjee 외를 참조하십시오., 2010c)에 의해 pretreated. <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="그림 2"/> 그림 2. AFEX - pretreated 옥수수 여물 (AFEX - CS), 알칼리 수소 과산화물 - pretreated 옥수수 여물 (AHP - CS) 또는 말린 AFEX - pretreated에서 Glc의 릴리스 11 효소의 최적 비율은 증류ERS &#39;곡식 (AFEX - DDG). 데이터 Banerjee 외에서 있습니다. (2010c). 막대의 상단 숫자는 바이오 매스 샘플의 총 Glc의 비율로 Glc의 산출됩니다. <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="그림 3"/> 그림 3. A. AFEX - pretreated DDG에서 Glc의 릴리스에 대한 네 상업 효소 준비의 최적 혼합을 생산 GENPLAT의 사용합니다. Multifect Xylanase의 작은 기여 (4 %)를 만들어 그러므로이 3 원 그림에서 생략되었습니다. 동일한 가수 분해 조건 (15 MG / G 글루칸 효소, 48 H, 50 ° C)에 따라 서로 다른 효소 치료를 AFEX - DDG에서 B. Glc의 수확량이 . &quot;4 - 구성 요소 상업은&quot;그림에 표시된 실험에서 결정된 비율을했다. 3A.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+microplate+assay+system+for+quantitative+evaluation+of+plant+cell+wall-degrading+enzyme+activity+of+fungal+culture+extracts.">An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.</a> Biotechnol. Bioeng. 102, 1033-1044 (2009).</li><li>Nagendran, S., Hallen-Adams, H. E., Paper, J. M., Aslam, N., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+genomic+potential+for+secreted+plant+cell-wall-degrading+enzymes+in+the+ectomycorrhizal+fungus+Amanita+bisporigera,+based+on+the+secretome+of+Trichoderma+reesei.">Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei.</a> Fung. Genet. Biol. 46, 427-435 (2009).</li><li>Rosgaard, L., Pedersen, S., Langston, J., Akerhielm, D., Cherry, J. R., Meyer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+minimal+Trichoderma+reesei+cellulase+mixtures+on+differently+pretreated+barley+straw+substrates.">Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated barley straw substrates.</a> Biotechnol. Prog. 23, 1270-1276 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

그것은 널리 효소의 비용을 절감하는 경제적인 리그노셀룰로오스성 에탄올 산업의 발전에 중요하다는 인식됩니다. 현재 사용 가능한 상용 효소 칵테일은 여​​러 단백질 (Nagendran 외., 2009)의 복잡하고 제대로 정의 혼합물이며, 그들은 주로 산성 - pretreated 옥수수 여물에 사용에 대한 적응입니다. 더 효소 칵테일의 발전을 촉진하기 위해서는 여러 실험실 효소 발견과 특성화에 대한 높은 처리량 플랫폼을 개발했습니다. .; 데커 동부 표준시 로봇 효소 및 바이오 매스 slurries의 분배, 실험의 통계적 설계, 및 / 또는 Glc의 자동 결정 및 Xyl (베를린 외, 2007 :이 영역에서 노력도 GENPLAT에서 찾은 다음 속성 중 하나 이상을 포함해야 알 2009;. 김 외, 1998;. 킹 외, 2009).. GENPLAT는 대부분 6 구성 요소에서 분석할 수있는 효소 혼합물의 복잡 가장 크게, 이러한 이전의 노력을 확장최근 작품 이상의 16 이전 연구 (Banerjee 외., 2010c). 완만한​​은 엔드 오버 엔드 회전에 의해 소화하는 동안 믹싱, 및 GLU와 Xyl의 자동 colorimetric 결정 GENPLAT의 추가 주요 기능은 분배하는 동안 정지 여물의 slurries을 유지할 수 있습니다 비드 혼합 챔버 사용 (패들 저수지)입니다.

사용 장비 및 소프트웨어 : <ol><li> 디자인 전문 소프트웨어, 버전 8.0 (합계 - 용이성, 주식 회사, 미네 아 폴리스, 미네소타) ( <a href="http://www.statease.com">http://www.statease.com</a> ) </li><li> Biomek FXP 실험실 자동화 워크 스테이션 (베크 만 - 보습 바로 앞에 달린 풀베는 날, 풀러턴, CA) </li><li> Biomek FXP 소프트웨어, 버전 3.3 (베크 만 - 보습 바로 앞에 달린 풀베는 날, 풀러턴, CA) </li><li> 이그 저수지 (모델 부사장 756C - 1P100, V &amp; P 과학 주식 회사, 샌디에고) </li><li> 접시, 96 - 깊이 (1.5 - ML) 잘 (Abgene, 피셔 과학, 엡손, 영국) </li><li> Pierceable capmats (미국 과학, Ocala, FL) </li><li> 회전 하이브 리다이 제이션 인큐베이터 (모델 5420, VWR) </li><li> 스윙 - 버킷 원심 분리기 (모델 5810R, Eppendorf, 함부르크, 독일) </li><li> 접시, Costar 96 - 웰 (Abgene, 피셔 과학, 엡손, 영국) </li><li> 접시, 384 자 (BD 팔콘, BD Biosciences, 산호세, CA) </li></ol> 시약은 사용 : <ol><li> Pichia pastoris의과 벡터 : 변형 X33, 그리고 pPICZ 및 pPICZα (Invitrogen, Carlsbad, CA) </li><li> T. reesei과 벡터 : 스트레인 QM9414 (농업 이용 연구 센터 국립 농업, 피오리아, 일리노이 미국학과) 및 pCB1004 - EV (Banerjee 외, 2010b). </li><li> Accellerase 1000 (많은 번호 1600844643) (Genencor, 주식 회사, 로체스터, NY) </li><li> 50 MM 나트륨 구연산 버퍼, 산도 4.8 </li><li> 테트라 사이클린 재고 : 10 밀리그램 / ML </li><li> Cycloheximide 재고 : 10 밀리그램 / ML </li><li> GOPOD 분석 키트, 카탈로그 K - GLUC (Megazyme, 브레이, 아일랜드) </li><li> 자일로 오스 분석 키트, 카탈로그 K - 자일로 오스 (Megazyme, 브레이, 아일랜드) </li></ol>

genplat: an automated platform for biomass enzyme discovery and cocktail optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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Watch this Scientific Journal Video about GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization at JoVE.com

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

GENPLAT (GLBRC 효소 플랫폼)는 바이오 매스 열화에 대한 효소 칵테일의 발견과 최적화를위한 자동화 플랫폼입니다. 이것은 여러 feedstocks 및 여러 구성 요소를 포함하는 효소의 혼합물에 적용할 수 있습니다.

GENPLAT : 바이오 매스의 효소 발견과 칵테일 최적화를위한 자동 플랫폼

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

genplat-an-automated-platform-for-biomass-enzyme-discovery-cocktail

Nanyang Technological University

Research

JoVE Journal

Bioengineering

15.4K 조회수.  Michigan State University. GENPLAT (GLBRC 효소 플랫폼)는 바이오 매스 열화에 대한 효소 칵테일의 발견과 최적화를위한 자동화 플랫폼입니다. 이것은 여러 feedstocks 및 여러 구성 요소를 포함하는 효소의 혼합물에 적용할 수 있습니다.

비디오: GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Biomaterials 높은 처리량 발견을위한 폴리머 Microarrays

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

연구

생체공학

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

효소 고정 및 안정화를위한 소수성 소금으로 바뀌었 Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

마이크로 스케일 세포 자극 실험을위한 다목적 자동화 플랫폼

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

미생물 Optogenetic 애플리케이션을위한 설계 및 자동화 일루미, 배양 및 샘플링 시스템의 구현

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

FR-4 구리 입히는 전극 플레이트 전도 Micropump에 대 한 성능 테스트 플랫폼

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

자동화 된 Microbioreactor 기술을 사용 하 여 분리 단백질 생산의 최적화를 위한 일반 프로토콜

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

단일 셀 게놈 시퀀싱 초고 처리량 미세 플랫폼

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Vivo에서 부분 간 엔지니어링을에서 위한 쥐를 기초로 새로운 수술 기법

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

세포질 기계 화학 Epigenetic 한정자를 리디렉션하여 억압 유전자 전사

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...