우리는 우리에게 박테리아 변종, plasmids, 필요한 프로토콜을 보내는 박사 마이클 카쉘을 (건강, 베데스다, MA, 미국의 국립 연구소) 감사합니다. 우리는 SpecraMax 플레이트 리더 사용에 대한 박사 존 글로버를 (생화학학과, 토론토 대학) 감사합니다. 영국 국립 과학 및 캐나다의 공학 연구 협의회 (NSERC) 대학원 장학금, 질병에 링크된 막 단백질의 구조 생물의 건강 연구 전략 훈련 프로그램의 캐나다 연구소, 그리고 토론토 오픈 원정대의 대학의받는 사람입니다. 이 작품은 WAH로 건강 연구의 캐나다 연구소 (걸레 - 67210)에서 부여에 의해 지원되었다.

1) 시약 및 장비 <ol><li> 우선, 다음과 같은 세 가지 솔루션을 준비 : 솔루션 1 ML 8 MM L - 라이신, 100 MM 나트륨이 구성되어있는 A - (N - Morpholino) ethanesulphonic 산성 (MES) 산도 6.5, 뉴클레오 티드는 염기 0.2 MM, 중 : diphosphate 구아 노신 (GDP), 구아 노신 삼인산 (GTP), 구아 노신 5&#39; - diphosphate, 3&#39; - diphosphate (ppGpp) 또는 5&#39; - 삼인산 구아 노신, 3&#39; - diphosphate (pppGpp), 0.1 MM 피리 독살 5&#39; - 인산 (PLP), 1 MM β - 메르 캅 토 에탄올 / (β - ME). 100 MM 나트륨 MES 산도 6.5, 0.1 MM PLP, 1 MM β - ME, 50 nm의 LdcI 구성되어 솔루션 B 1 ML. 스나이더 외에 설명된대로 LdcI이 정화되었다. 6 Kanjee 외. 14. 솔루션 B와 동일하지만 LdcI를 포함하지 않는 솔루션 C 1 ML. </li><li> 멀티 채널 피펫을 사용하여 96 - 웰 폴리스티렌 플레이트의 각 우물에 1 M의 나트륨 탄산염 (10.6 g/100 ML)과 나누어지는 50 μL로 구성된 중지 솔루션의 100 ML를 준비합니다. 접시에 물을 30 μL를 추가하고 다음 커버. 물 수용량의 합계가 추가되고 효소 반응 동안 추출한 시료의 부피가 50 μL를 동일해야합니다. 이 경우에는, 효소 반응 시료 20 μL가 삭제됩니다. </li><li> 알루미늄 호일에 4,706 μL 물, 랩 5 %를 294 μL (W / V) TNBS 주식을 diluting 10 MM에서 TNBS 솔루션의 5 ML을 준비하고 얼음 계속. </li><li> Eppendorf의 온​​도를 평형 플러스 37 ° C. 온도 조절기 플러스는 6 열 24 우물이 있습니다. 배선도를 그림 2를 참조하십시오. (열 당 하나) 테스트 될 뉴클레오 티드의 신분을 나타내는 라벨 1.5 ML Eppendorf 튜브 : GTP (열), GDP (열 B), ppGpp (C 열), pppGpp (열 D), 아니 뉴클레오 티드 (열 E ), 그리고 단백질 샘플 (열 F). </li><li> 각각의 다른 행을 팁 4 열로 총을 제공 생략되는 등 200 μL 팁 랙 몇 상자. 이것은 효소 분석하는 동안 온도 플러스 heatblock과 함께 멀티 채널 피펫의 사용을 위해 필요합니다. </li><li> 42 디지털 Heatblock (VWR)을 평형 ° C. </li><li> 냉각 얼음에 96 - 웰 2.0 ML의 폴리 프로필렌 플레이트를 놓습니다. </li></ol> 2) LdcI 분석 <ol><li> 솔루션 나누어지는 50 μL Eppendorf 온도 조절기의 다섯 기둥의 각 1.5 ML Eppendorf 튜브에 적절한 염기와 함께. </li><li> 열 F 세 튜브에 솔루션 B의 330 μL를 추가하고 열 F.에서 최종 튜브에 솔루션 C (노 단백질 제어)의 330 μL를 추가 </li><li> 37 솔루션을 평형 ° C를 5 분 동안. 팁 : 5 분 후에 다음 사용할 수있는 멀티채널 피펫 방해에서 그들을 방지하기 위해 가열 블록의 중심에있는 튜브의 뚜껑을 잘라. </li><li> 열 AE의 튜브의 각으로 열 F에있는 튜브에서 5-50 VWR μL 멀티 채널 피펫, 이전 50 μL를 사용하여. 마자 첫 번째 튜브가 혼합으로 타이머를 시작합니다. </li><li> 2, 4, 6 분 멀티 채널 피펫을 사용하여 샘​​플을 20 μL를 제거하고 중지 솔루션에 추가할 수 있습니다. </li><li> 참고 : 중지 솔루션 샘플 플라스틱 포장으로 덮여 이후 처리를 위해 -20 ° C에 냉동 수 있습니다. 접시는 TNBS로 반응하기 전에 상온에서 해동해야합니다. </li></ol> 3) TNBS 반응 및 색상 개발 <ol><li> 육분에 대한 다음 멀티 채널 피펫 42에서 품어 ° C를 사용 중지 솔루션 10 MM의 TNBS 솔루션 50 μL를 추가합니다. TNBS은 라이신과 cadaverine와 반응으로 솔루션은 어두운 노란색 / 오렌지 색상을 설정합니다. </li><li> 육분 후, 반응을 느리게하기 위해 얼음 접시를 냉각. </li><li> 얼음 냉각되었다 2 ML 딥 웰 플레이트에 VWR 20-200 μL 다중 채널 피펫과 장소를 사용하여 샘​​플을 100 μL를 제거합니다. fumehood에 얼음 양동이를 전송합니다. </li><li> 잘 HandyStep (브랜드) 반복 pipettor와 12.5 ML 피펫 팁 (Plastibrand)를 사용하여 각 톨루엔 500 μL를 추가합니다. </li><li> kimwipe를 사용하여 초과 톨루엔을 닦아 및 포장 테이프의 스트립과 접시를 커버. 좋은 도장을 얻기 위해 단단히 눌러해야합니다. 평면 뚜껑과 96 - 웰 플레이트를 커버하고 1 분 30 초 동안 적극적으로 흔들. </li><li> 솔루션 5 분 정착을 둡니다. TNP - 라이신은 물 (그림 1 참조) 가용성 유지하면서 TNP - cadaverine은 상단 톨루엔 단계에 지금있다. </li><li> 톨루엔은 독서를위한 96 - 웰 플레이트에 20-200 μL 멀티 채널 피펫을 사용하여 200 μL를 제거합니다. 참고 : 제거하는 각 샘플이 명확하고 아래 수성 단계 중 하나가없는 것을 확인할 수 있는지 확인합니다. 참고 : 톨루엔의 멀티 채널 피펫의 손상을 방지하기 위해 사용 장벽 팁. </li><li> SpectraMax 340 플레이트 판독기에서 340 nm의 흡광도에서를 읽으십시오. </li><li> 석영 판을 청소하려면 물로 톨루엔을 씻어 대형의 석영 판을 배치펌 후드에 유리 트레이. 70% (V / V) 질산의 산성의 7시 3분 혼합 100 ML 이상 붓고 : 95 % (V / V) 에탄올과 두 번째 유리 트레이와 석영 판을 커버. 경고 : 반응이 높은 발열과 때로는 폭발적인 마모 적절한 보호되고 퓸 후드 허리띠가 낮은 6 &quot;레벨로 설정되어 있는지 확인 냉각 후 물을 질산을 씻어 후 95 % 에탄올.. </li></ol> 4) 대표 결과 1) Cadaverine 표준 곡선 (그림 3) 분석은 같은 L - 라이신 또는 LdcI없이 설명하지만, 수행되었으며 cadaverine 대신에 다양한 농도는 경험적으로 분석의 선형 범위를 결정하는 데 사용되었습니다. 분석은 cadaverine의 ~ 22 nmoles (그림 3)에 해당, 0.25의 OD 340 선형했다. LdcI 2) 활동 (그림 4) 혼자 LdcI의 활동은 153.5 (± 18.1) nmoles cadaverine 분 - 1 산도 6.5에서 μg LdcI - 1. 것으로 결정되었다 LdcI의 활동은 100 μm의 GTP 또는 GDP의 존재에 영향을받지 않지만 강하게 pppGpp 및 ppGpp (그림 4)의 존재 (&gt; 10 배) 저해합니다. <img src="/files/ftp_upload/2094/2094fig1.jpg" alt="그림 1" /> 그림 1. LdcI 반응의 개략도 다이어그램. CO 2와 cadaverine를 생성하는 L - 라이신의 decarboxylation 반응은 N, N&#39; - bistrinitrophenylcadaverine (TNP - cadaverine)과 N, N을 생성뿐만 아니라 높은 산도에서 TNBS로 후속 반응으로 보여집니다 &#39;- bistrinitrophenyllysine (TNP - 라이신). 판 동부 표준시 al.11에 따라 <img src="/files/ftp_upload/2094/2094fig2.jpg" alt="그림 2" /> 그림 2. Eppendorf 온도 조절기의 설정은. 24 잘 Eppendorf 온도 조절기에있는 샘플의 배치는 분석의 단계에 대한 설명 (굵게 표시)와 함께 표시됩니다. 화살표는 프로토콜에 따라 반응 솔루션의 전송을 나타냅니다. 96 - 웰 플레이트에 중지 솔루션에 대한 반응 솔루션의 제거도 표시됩니다. <img src="/files/ftp_upload/2094/2094fig3.jpg" alt="그림 3" /> 그림 3. Cadaverine 표준 곡선은. cadaverine의 nmoles 비교 340 nm의 흡광도에서의 플롯이 표시됩니다. 오차 막대는 적어도 세 독립 측정의 표준 편차를 나타냅니다. 가장 적합의 라인도 표시와 0.996의 R2를 가지고있다. <img src="/files/ftp_upload/2094/2094fig4.jpg" alt="그림 4" /> 그림 4. LdcI 분석 결과. LdcI 활동의 속도의 줄거리는 100 μm의 GTP, GDP, pppGpp, ppGpp의 존재와 염기의 부재에 표시됩니다 (nmoles cadaverine에서 분 - 1 μg - 1 LdcI을 생산). 엄격한 응답 세포핵 (P) ppGpp 크게 억제 LdcI 활동 할 수 있습니다. 오차 막대는 적어도 6 독립 측정의 표준 편차를 나타냅니다.

<ol>
	<li>Gale, E. F., Epps, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+pH+of+the+medium+during+growth+on+the+enzymic+activities+of+bacteria+(Escherichia+coli+and+Micrococcus+lysodeikticus)+and+the+biological+significance+of+the+changes+produced.">The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced.</a> Biochem J. 36, 600-618 (1942).</li><li>Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+acid+resistance:+tales+of+an+amateur+acidophile.">Escherichia coli acid resistance: tales of an amateur acidophile.</a> Nat Rev Microbiol. 2, 898-907 (2004).</li><li>Castanie-Cornet, M. P., Penfound, T. A., Smith, D., Elliott, J. F., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+acid+resistance+in+Escherichia+coli.">Control of acid resistance in Escherichia coli.</a> J Bacteriol. 181, 3525-3535 (1999).</li><li>Iyer, R., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine-agmatine+antiporter+in+extreme+acid+resistance+in+Escherichia+coli.">Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli.</a> J Bacteriol. 185, 6556-6561 (2003).</li><li>Sabo, D. L., Boeker, E. A., Byers, B., Waron, H., Fischer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+physical+properties+of+inducible+Escherichia+coli+lysine+decarboxylase.">Purification and physical properties of inducible Escherichia coli lysine decarboxylase.</a> Biochemistry. 13, 662-670 (1974).</li><li>Snider, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+distinctive+complex+between+the+inducible+bacterial+lysine+decarboxylase+and+a+novel+AAA++ATPase.">Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase.</a> J Biol Chem. 281, 1532-1546 (2006).</li><li>Kashiwagi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+the+genes+for+putrescine+transport+protein+and+ornithine+decarboxylase+at+16+min+on+Escherichia+coli+chromosome.">Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome.</a> J Biol Chem. 266, 20922-20927 (1991).</li><li>Schneider, G., Kack, H., Lindqvist, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+manifold+of+vitamin+B6+dependent+enzymes.">The manifold of vitamin B6 dependent enzymes.</a> Structure. 8, 1-6 (2000).</li><li>Cashel, M., Gentry, D. R., Hernandez, V. J., Vinella, D., Curtiss, R., Neidhardt, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Escherichia coli and Salmonella : cellular and molecular biology. , 1458-1496 (1996).</li><li>Magnusson, L. U., Farewell, A., Nystrom, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp:+a+global+regulator+in+Escherichia+coli.">ppGpp: a global regulator in Escherichia coli.</a> Trends in Microbiology. 13, 236-242 (2005).</li><li>Phan, A. P., Ngo, T. T., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+assay+for+lysine+decarboxylase.">Spectrophotometric assay for lysine decarboxylase.</a> Anal Biochem. 120, 193-197 (1982).</li><li>Park, Y. K., Bearson, B., Bang, S. H., Bang, I. S., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+pH+crisis,+lysine+decarboxylase+and+the+acid+tolerance+response+of+Salmonella+typhimurium.">Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium.</a> Mol Microbiol. 20, 605-611 (1996).</li><li>Merrell, D. S., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cadA+gene+of+Vibrio+cholerae+is+induced+during+infection+and+plays+a+role+in+acid+tolerance.">The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance.</a> Mol Microbiol. 34, 836-849 (1999).</li><li>Kanjee, U., Gutsche, I., Alexopoulos, E., Zhao, B., Bakkouri, M. E. l., Thibault, G., Liu, K., Ramachandran, S., Snider, J., Pai, E. F., Houry, W. A., A, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+between+the+Bacterial+Acid+Stress+and+Stringent+Responses+Revealed+by+the+Structure+of+the+Inducible+Lysine+Decarboxylase.">Linkage between the Bacterial Acid Stress and Stringent Responses Revealed by the Structure of the Inducible Lysine Decarboxylase.</a> EMBO Journal. 30, 931-944 (2011).</li></ol>

관심 없음 충돌 선언하지 않습니다.

리진 탈카복실화효소 분석에서 TNBS은 TNP - 라이신과 TNP - cadaverine 부가물 (그림 1)을 형성 L - 라이신과 cadaverine의 기본 아민과 반응입니다. 카르복실산 그룹을 부족 TNP - cadaverine은, 톨루엔 11로 분할 할 수있는 동안 TNP - 라이신의 카르복실산 그룹의 존재로 인해,이 adduct은 물에 용해 남아 있습니다. 분석의이 유형은 카르복실산 그룹의 손실이 반응 중에 발생하는 아미노산의 다른 유형에 대?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,4,6-trinitrobenzensulfonic acid </td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2297</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mM pyridoxal 5'-phosphate (PLP)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P9255</td>
<td>&nbsp;</td>
<tr>
<td>Cadaverine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>D22606</td>
<td>&nbsp;</td>
<tr>
<td>96 well polystyrene plates</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>96-well quartz plate</td>
<td>&nbsp;</td>
<td>Hellma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>VWR Digital Heatblock</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ThermoStat Plus</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2.0 mL 96-well polypropylene plates</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>P-DW-20-C</td>
<td>&nbsp;</td>
<tr>
<td>Handy Step Repeat Pipettor</td>
<td>&nbsp;</td>
<td>Brand</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>12.5 mL Repeat Pipettor Tips</td>
<td>&nbsp;</td>
<td>Brand (Plastibrand)</td>
<td>702378</td>
<td>&nbsp;</td>
<tr>
<td>SpectraMax 340PC Plate Reader</td>
<td>&nbsp;</td>
<td>SpectraMax</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an assay for measuring the activity of escherichia coli inducible lysine decarboxyase

1, 매우 낮은로 산도가 가을 수있는 포유류의 위장을 통해 통로를 포함하여 극단적인 산성 스트레스를 이겨낼 수있다 - 대장균은 산도 값 (9 산도 5) 다양한 범위에서 성장이 가능합니다 장용 박테리아입니다 로 산도 2월 1일부터 2일까지. 산성 산도의 생존, E. 이러한 광범를 활성화하려면 대장균은 양성자 종속적인 방식으로 decarboxylate 자신의 기판 아미노산이 때문에 내부 산도를 높여 그 네 가지 inducible 아미노산 decarboxylases을 가지고 있습니다. decarboxylases은 glutamic 산성 decarboxylases 가다와 GadB 3, 아르기닌 탈카복실화효소 AdiA 4 리진 탈카복실화효소 LdcI 5, 6 및 ornithine 탈카복실화효소 SpeF 7 포함되어 있습니다. 이러한 효소의 모든 공동 요소 8로 피리 독살 - 5&#39; - phospate 활용과 신선한 기판 2 교환에 외부 매체 decarboxylation 제품을 제거 - 멤브레인 내부 기판 - 제품 antiporters와 함께 작동합니다. LdcI의 경우, 라이신 - cadaverine antiporter은 CadB라고합니다. 최근, 우리는 2.0 Å에 LdcI의 X - 선 결정 구조를 결정하고, 우리는 LdcI 엄격한 응답 조절기 구아 노신 5&#39; - diphosphate, 3&#39; - diphosphate (ppGpp)가 14 바운드 소설 작은 분자를 발견했다. 기하 급수적으로 성장하는 세포가 영양 부족이나 기타 스트레스 9 숫자 중 하나가 발생할 때 엄격한 응답이 발생합니다. 그 결과, 세포는 고정 위상 성장 10 기하 급수적인 성장의 변화에 culminating 신호 계단식으로 연결 ppGpp을 생산합니다. 우리는 ppGpp는 LdcI 14 특정 억제제는 것을 증명하고있다. 우리가 판 외 개발한 분석에서 수정된 라이신 탈카복실화효소 분석을 설명합니다. 11, 우리가 LdcI의 활동과 그 활동에 pppGpp / ppGpp의 효과를 결정하는 데 사용했다고. LdcI의 decarboxylation 반응은 L - 라이신의 α - 카르복시 그룹을 제거하고 이산화탄소 및 polyamine cadaverine (1.5 - diaminopentane) 5을 생산하고 있습니다. L - 라이신과 cadaverine는 각각 N, N&#39; - bistrinitrophenylcadaverine (TNP - cadaverine)와 N, N&#39; - bistrinitrophenyllysine (TNP - 라이신)을 생성하기 위해 높은 산도에서 2,4,6 - trinitrobenzensulfonic 산성 (TNBS)와 반응 수 있습니다 11. TNP - cadaverine은 후자가 (그림 1 참조)는 아니지만, 전 그런 톨루엔과 같은 유기 용제에 용해와 같이 TNP - 라이신으로부터 분리 수 있습니다. 분석의 선형 범위는 경험적으로 정화 cadaverine를 사용하여 결정되었다.

Watch this Scientific Journal Video about An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase at JoVE.com

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

inducible 리진 탈카복실화효소의 활동은 톨루엔에 차등 용해도를 부가물을 형성 2,4,6 - trinitrobenzensulfonic의 소산으로 기판 L - 라이신과 제품 cadaverine을 반응에 의해 모니터링됩니다.

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 ...

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Biology

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

14.1K 조회수.  University of Toronto. inducible 리진 탈카복실화효소의 활동은 톨루엔에 차등 용해도를 부가물을 형성 2,4,6 - trinitrobenzensulfonic의 소산으로 기판 L - 라이신과 제품 cadaverine을 반응에 의해 모니터링됩니다.

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

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

연구

생물학

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.

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 ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

라이신 Demethylase 활동의 양적 측정을위한 MassSQUIRM의 응용

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

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 ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

구현을위한 프로토콜<em&gt; 대장균</em합성 생물학&gt;을 바탕으로 TX-TL의 무 세포 발현 시스템

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

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. ...

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 ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

를 사용하여 모델 단백질로 비정규 아미노산의 잔류 특정 법인 설립<em&gt; 대장균</em&gt; 휴대없는 전사 번역 시스템

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...