수치를 다시 인쇄하는 스프링과 엘스 비어에 의한 권한은 크게 인정 받고 있습니다.

E. 1. 분리 대장균 공동 형질 전환 <ol><li> 해당 E.의 전기 유능한 세포를 준비 대장균 균주를 변환 할 수 있습니다. LB 배지 1 ㎖ (트립 톤, 효모 추출물, (10)에서의 NaCl, 5, 10g / 각각 L,) 선택의 변형의 단일 식민지, 그리고로 전송 180 rpm으로 진탕 조건에서 37 ° C에서 배양한다. 사전 문화 (1) 희석 : 500 신선한 LB 배지 m​​l의 25에서 37 ° C에서 문화를 품다. </li><li> 로그 상 (0.6 OD)에서 20 분 동안 5,000 XG)에서 세포 현탁액을 원심 분리하고 원래 문화 볼륨의 절반에 10 %, v / V를 얼음처럼 차가운 멸균 글리세롤 물에 펠렛을 재현 탁. 마다 부유 양을 절반으로,이 단계를 4 회 반복한다. 마지막으로, 글리세롤 / 물에 펠렛을 재현 탁하고 50 ㎕의 분취 액에 세포 현탁액을 나눈다. 6개월에 -80 ° C 최대의 분량을 저장합니다. </li><li> 0.5 mM의 EDTA 보충 멸균에서 원하는 플라스미드를 녹여. IC에 해동전자 전기 - 반응성 세포의 분취 량과 벡터의 적절한 양 (2.5-5 NG)와 혼합한다. 전기에 적합한 0.1 cm 큐벳에 혼합물을 분배하고, 1.8 kV의 적용. </li><li> 즉시 SOC 배지 1 ㎖로 일렉트로 세포를 트랜스퍼 (LB 배지 w 0.2 % / V 포도당 된 10 mM의 MgCl 2, 2.5 mM의 KCl을 보충), 진탕하에 1 시간 동안 배양하고, 마지막으로 함유하는 페트리 접시에 100 μL 씩 전송 적절한 항생제 LB 한천. 37 ° C에서 O / N을 품어. </li><li> 페트리 접시에 하나의 식민지를 질주하여 형질 전환 체를 정화. </li><li> 기본 형질 전환 체의 전기 유능한 세포를 준비하고 반복 더 플라스미드로 변환하는 1.1-1.5 단계를 반복합니다. </li><li> 공동 형질 전환 체의 글리세롤 주식을 준비합니다. 적절한 항생제로 보충 LB에서 하나의 식민지로 이동 진탕 37 ° C에서 배양하고, 로그 상 (0.6 OD) 원심 분리기 20 분 5,000 XG에 세포 현탁액에서. R중간 15 % v / V를 글리세롤을 포함하는 LB-항생제의 펠렛을 esuspend. 분량 씩 분배와에 저장 - 80 ° C. </li></ol> E.의 α와 ε 소단위 2. 공동 식 대장균 DNA 중합 효소 III <ol><li> 멸균 된 루프 적절한 변형 글리세롤 스톡 소량 전송 LB 배지 및 항생제를 함유하는 페트리 접시에 (예 TOP10 / PGOOD-ε243 및 TOP10 / PGOOD-ε243-θ / pBADα1160를,도 1 참조). 세포가 방울 세포 현탁액 페트리 접시 위에 건조하고, 행진하자. 37 ° CO / N에 품어. </li><li> 멸균 된 이쑤시개, LB-항생제 매체의 1 ml의 단일 콜로니를 사용하여 전송. 37 ° C에서 8 시간을 품어. 신선한 매체 (500)를 15 시간 동안 30 ° C에서 부화 : 사전 문화 (1) 희석. </li><li> IPTG, 아라비 노스, 또는 아라비 노스와 IPTG, 1 mM의 각을 추가합니다. 2.5 시간 동안 30 ° C에서 품어. 세포를 수집하고, -20 ° C에서 알약을 저장합니다. </li><li> 용해 완충액 (50 mM Tris-HCl, 50 mM의 염화나트륨, 1 mM의 EDTA, 2.5 mM의 디티 오 트레이 톨, 1​​mM의 페닐 메틸 설 포닐 플루오 라이드 (PMSF), pH가 8)에 재현 탁하고 얼음에 녹여 펠릿. 조심스럽게 차가운 유리 포터와 세포 현탁액을 균질화. </li><li> 15 초 냉각 간격 (4 사이클) 다음 15 초 15 W,에서 초음파 처리. </li><li> 가용성 획분을 회수하는 4 ° C에서 20 분 동안 10,000 XG에서 전체 단백질 추출물을 원심 분리기. </li><li> SDS-PAGE (아크릴 아미드 12.5 %) 각각의 가용성 단백질 추출물의 분액에 의해 분석한다. 이를 위해, H 2 O 60 ㎕의 로딩 완충액 (250 mM 트리스 - 염산 pH를 20 μl를 함유하는 에펜 도르프 튜브에 각각 가용성 단백질 추출물 20 μL를 전송 6.8, 10 밀리미터 β 머 캅토 에탄올, 10 % (w / v)의 SDS, 50 % (v / v) 글리세롤, 0.25 % 청색 (w / v)의 브로 모 페놀)과 5 분 동안 끓인다. 로드 각 샘플의 18 μl의 약 1.5 시간 동안 140 V의 전기를 수행합니다. </li></ol> Deinococ의 α 서브 유닛 3. 공동 식기분이야 radiodurans DNA 중합 효소 III와 ε E. 소단위 대장균 DNA 중합 효소 III <ol><li> E.의 사전 문화를 준비 대장균의 TOP10 / 박사를 pBAD-α / LB-암피실린 - 테트라 사이클린 배지 1 ㎖에 PGOOD-ε243 37 ° C에서 8 시간을 품어. 새로운 배지에서 1,000 37 ° C에서 O / N을 품어 : 1 희석. </li><li> 1 희석 : 신선한 매체의 100 (200)에 ㎖를, 다음 아라비 노스와 IPTG, 1 mM의 각각 3 시간 동안 유도, 3 시간 동안 37 ° C에서 품어. 세포를 수확하고, -20 ° C에서 펠렛을 저장합니다. </li><li> 50mM 트리스 - 염산 pH를 8, 150 mM의 염화나트륨, 1 mM의 EDTA, 1mM의 PMSF에 펠렛을 재현 탁. 균질화 2.4 및 2.5에 기술 된 바와 같이 세포를 방해. </li><li> 즉시 세포를 20 분 동안 10,000 XG에 원심 해물. 펠렛을 폐기하십시오. 종이 필터의 3 층을 구비 한 흡인 여과기와 상청액을 필터. 부드러운 진공을 적용합니다. 과도한 거품을 방지하기 위해 여과 동안 얼음에 진공 삼각 플라스크를 유지합니다. </li><li> 브래드 포드 (14)에 따라 단백질 농도를 결정합니다. </li></ol> 4. 젤 여과 크로마토 그래피 <ol><li> 50 mM 트리스 - 염산, 150 mM의 염화나트륨, 1 mM의 EDTA, pH를 컬럼으로 8로드 가용성 단백질 추출물과 물 재킷 16x70 (200) 겔 여과 열 평형. 최적 해상도를 들어, 1 ml의 시료 루프를 사용합니다. / 분 0.6 ml의에서 크로마토 그래피를 수행합니다. 에 걸쳐 4 ° C에서 열 온도를 유지합니다. </li><li> 0.9 ml의 분수를 수집하고 SDS-PAGE에 의해을 분석 할 수 있습니다. 전송 H 2 O 60 μL 및 로딩 완충액 (250 mM 트리스 - 염산 pH를 6.8, 10 밀리미터 β 머 캅토 에탄올, 10 % (/ V) SDS w, 50 %의 20 μl를 함유하는 에펜 도르프 튜브에 관련된 각 분획의 20 μL ( v / v)의 글리세롤, 0.25 % (파란색 w / v)의 브로 모 페놀) 및 5 분 동안 끓인다. 로드 각 샘플의 18 μl의 약 1.5 시간 동안 140 V의 전기를 수행합니다. </li><li> 3&#39;-5 &#39;엑소 뉴 클레아 제 및 각 분획의 DNA 중합 효소의 활성을 96 우리하여 결정LL 마이크로. 기판 (15)로 티미 딘 5&#39;- 모노 인산 (P) 니트로 에스테르 (p-TMP의 NP)을 이용하여 엑소 뉴 클레아 제 활성을 분석. PPX (Pyrophosphatase, 퓨린 뉴 클레오 사이드 포스 포 릴라 제, 크 산틴 옥시 다제), 효소 결합 분석 16을 사용하여 DNA 폴리머 라 아제 활성을 예측 2- (4- 요오도 페닐) -3- (4- 니트로 페닐) -5- 페닐 -2H-테트라 졸륨 클로라이드 (INT) 전자 수용체 16. </li></ol> 인구 E.의 야생 형 ε 서브 유닛을 공동 발현 5. 돌연변이 분석 대장균 DNA 중합 효소 III 및 변이원성 εD12A 변형 <ol><li> E.의 단일 식민지로 이동 pBAD-ε과 LB-항생제 배지 1 ㎖에 pGOOD1-εD12A 11 벡터를 포함하는 대장균 TOP10. 37 ° C에서 O / N을 품어. </li><li> (아라비 노스, IPTG, 또는 아라비 노스 및 IPTG, 1 mM의 각각)을 적당한 유도제를 추가 한 새로운 배지 (10 ㎖)을 함유하는 플라스크에 250 3 37 ° C에서 배양한다 : 예비 배양 한 희석8 시간 동안. 병렬이 아닌 유도 문화에서 준비합니다. </li><li> 1 ml의 일정량을 수집 한 후 1 희석 : 신선한 매체 (10 ㎖)를 포함하는 새로운 플라스크에 500을 적절한 유도 보충 여부. 37 ° C에서 O / N을 품어. </li><li> 반복은 1 ml의 5.2 및 5.3 및 수집 분량 씩 단계를 반복합니다. </li><li> 세대의 수는 각 문화에서 발생 확인합니다. LB 플레이트, 접종 및 문화의 적절한 희석액 100 ㎕에 전송합니다. 37 ° C에서 O / N을 품어. LB 플레이트상에서 콜로니를 카운트하고, 성장 (C 로그)의 단부에 존재 균액 (I 로그)와 배양 세포 수의 로그를 계산한다. 세대의 수를 확인하려면 수식을 사용하십시오 (C 로그 - I 로그인) /0.301을. </li><li> 20 분 동안 5,000 XG 원심 분리기 및 50 mM 트리스 - 염산 pH를 7.6, 50 mM의 NaCl을 1 ml의 세포를 재현 탁. 세포를 Permeabilize 하시려면하려면 20 초 동안 클로로포름과 소용돌이 ~ 3 방울을 추가합니다. </li><li> β- 결정기판으로서 P 개의 -nitrophenyl-β-D 글루 코피 라노 사이드 (PNPGluc)를 사용하여 96- 웰 마이크로 플레이트의 각 분취 량 글루코시다 제 활성. 각 웰에 투과 가능 세포를 100 ㎕)과 기판 (100 ㎕ (16 ㎎ / H 2 O ㎖의 원액)를 추가합니다. 우물에 공기 방울을 피하기 위해주의하십시오. 마이크로 플레이트 리더 및 적절한 필터를 사용하여 420 nm에서 흡광도를 읽는다. </li></ol>

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The authors declare that they have no competing financial interests.

단백질은 그 차 구조 배좌 (25)의 제한된 수의 제한되지 않은 영역을 갖춘, 무질서 본질적 일 수있다. 이들 무질서 화 된 단백질은 통상 25 응집 경향이며, 그들의 분리 및 특성 규명은 어려운 작업을 나타낼 수도있다. E.의 ε 소단위 대장균 DNA 중합 효소 III는 두 가지 영역, 즉 (26, 27)를 특징 : I) N-터 도메인, θ 서브 유닛을 바인딩에 3&#39;-5 &#39;엑소 뉴 클레아 제 ...

과발현 기술의 도입은 낮은 카피 수 효소의 생화학 적 연구와 약리학 적 활성 단백질 (예를 들면, 인슐린)의 산업 생산를 부스팅. 이러한 기술의 출현 때문에 상당한 진보는 재조합 단백질의 수율 및 품질을 증가시키기 위해 달성되어왔다. 또한, 원핵 및 진핵 3 1,2- 과발현 시스템은 단백질 공학, 즉, 대장균 &quot;일꾼&quot;유용한 대안을 제공 년에 걸쳐 개발되었다. E. 대안 플랫폼 특히, 가용성 대장균 재조합 펩타이드 또는 번역 후 변형을 함유하는 단백질의 제조되었다. 그러나, 언급되어야 E. 대장균은 여전히 재조합 단백질 생산을위한 선택의 유기체를 나타냅니다. 이 가장 관련성이있을 수있는 여러 요소 중에서,에 기인고려 : ⅰ) 과발현 시스템 (발현 벡터 및 균주 확실히 다수의 가능 여부)을위한 E. 대장균 1,2; E. II의) 짧은 생성 시간 및 높은 수율의 바이오 매스, 풍부하고 합성 다양한 미디어에서 대장균; ⅲ) 생화학에서이 미생물의 유전자 수준에서 어느 손쉬운 조작; ⅳ) 4 독성 단백질의 생산이 가능한 균주의 분리; V) 인구 레벨 5,6에 균일 유도를 갖춘 균주의 건설. 또한, 최근에 E. 그 생산에 적합한 발현 시스템을 도시 한 번역 후 변형은 단백질의 대장균이 고안을 구성 할 수있다. 현재, 단백질의 과발현은 주로 그의 hypersynthesis 적절한 플라스미드에 클로닝 한 유전자와 함께 수행 될 수 단량체 또는 호모 올리고머 단백질을 얻기 위해 사용된다. 그러나 관심은 최근에 있었다E. 건설에게 지급 콜라이 단백질의 공동 발현 시스템, 헤테로 올리고머 성 복합체 (2)의 생체 내, 생산 도전. 흥미롭게도, 단백질의 공동 표현의 초기 실험은 시아 노 박테리아 리불 로스 -1,5- 비스 포스페이트 카르 복실 / 시게나 제 7,8의 크고 작은 소단위의 간 종 조립 및 HIV-1의 절단 및 전체 길이 형태의 관계를 해결 효소 구 역. 이러한 선구적인 연구는 단백질의 공동 발현이 체외 재구성 전통에 대한 강력한 대안을 나타낸다는 것을 보여 주었다. E.에서 또한, 단백질의 공동 발현 대장균은 천연 아미노산이 함유 단백질을 얻기 위해, 번역 후 변형이 담지 다른 단백질을 생산하고, 과발현 된 막 단백질 (2)의 수율을 증가시키기 위해 사용되었다. 또한, 단백질과 같은 공구의 공동 발현의 전위를 부여하는 E. 대장균 경쟁단백질 분비 후부 활성 조사 2 중입니다. E. 단백질의 공동 표현의 두 가지 주요 전략 대장균 추구 할 수 있습니다 다른 유전자를 호스팅 할 하나의 플라스미드의 사용이 과발현되는 I); ⅱ) 단일 셀에서 여러 플라스미드를 사용하는 목적 단백질의 공동 발현한다. 탠덤 프로모터 / 운영자 특정 원소를 함유하는 플라스미드가 공동 - 식 (10)에 대해 구축되었다하더라도 첫 번째 경우, 플라스미드의 선택을위한 기준은, 전통적인 단일 단백질 과발현 실험의 것과 다르지 않다. 첫 번째 방법은 따라서 매우 간단합니다. 그러나, 다른 단백질을 공동 발현하는 하나의 플라스미드를 사용하는이 두 가지 문제에 직면 언급한다 : ⅰ) 공동 발현 된 단백질의 수를 제한 호스팅 유전자 수가 벡터 증가의 분자량; ⅱ) 다수의 단일 유전자 프로모터의 제어하에 복제 될 때, 극성을 수 decreas원위 프로모터 유전자 발현 전자. E. 이중에서 단일 또는 다수의 플라스미드를 사용 대장균 세포 따라서 플라스미드의 자격이 조합에 제약을 부과, 선택의 벡터의 호환성을 수행 할 수 있습니다. 그러나,이 두 번째 공동 발현 전략은 벡터의 분자량을 포함하는 장점을 특징으로하고, 극성을 제한한다. 우리는 최근 공동 발현 플라스미드 (11) 유전자의 셔틀 링을 용이하게하기위한 단백질의 공동 발현 시스템을 구축 하였다. 특히, 우리 PGOOD 벡터 시리즈 지어진 중요한 특징은 어느이다 : ⅰ) 복제 p15A 오리진, 상업적 벡터 (예, pBAD 시리즈 12) ColE1 기원을 함유 함께 PGOOD 플라스미드의 호환성을 제공하는 단계; ⅱ) 테트라 사이클린 내성 카세트; ⅲ) 락의 존재는 규제 요소, 즉, 프로모터 연산자 (O 1) 부부와의 lacI을 - 유래 </em&gt; Q 유전자. 적절한 pBAD-PGOOD 커플을 사용하여, 우리는 E.의 촉매 코어를 과발현 할 수 있었다 세 개의 다른 소단위으로 구성 대장균 DNA 폴리머 라제 III, 즉, α (5&#39;-3 &#39;중합 효소), ε (3&#39;-5&#39;엑소 뉴 클레아 제) 및 (안정화 ε) 13 θ. 특히, αεθ 복합체의 공동 발현 E.에 더하여에 엄격히 의존 시연 IPTG와 아라비 모두 대장균 배양액 PGOOD 및 pBAD 각각 (도 1a)에서 트리거링 과발현. 본 보고서에서, 우리는 단백질의 공동 발현이 효율적으로 단백질 복잡한 서브 유닛의 가난한 용해도에 연결된 어려움을 해결할 수있는 방법을 보여줍니다. 또한, 어떻게 생체 단백질 보완 테스트에서 행할 수 보여, 우리는 마침내 E. 튜닝 주파수 변이 단백질로 공동 발현의 사용에 대한 보고서 안부내가. 이를 위해, 우리는 각각의 사례 연구의 관련 사례를 설명하기에 적합 PGOOD-pBAD 커플을 사용했다.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agar</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1296</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">288306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">EDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">INT</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">I8377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">I5502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Arabinose</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl2</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNP-gluc</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">N7006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pNP-TMP</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T0251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetracyclin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma base&#160;</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptone</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">95039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast extract</td>
			<td style="width:125px;">Fluka</td>
			<td style="width:119px;">70161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acrylamide solution 30%</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0158</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium persolphate</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0700</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0718</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0302</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0800</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0719</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cuvettes 0.1 cm</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">1652089</td>
			<td>For electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EQUIPMENT</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge 5415R</td>
			<td style="width:125px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Allegra 21R</td>
			<td style="width:125px;">Beckman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chromatography apparatus GradiFrac</td>
			<td style="width:125px;">Pharmacia Biotech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Pulser II electroporation</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Reader 550</td>
			<td style="width:125px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MiniProtean 3 cell</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Power Supply</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator 3000</td>
			<td style="width:125px;">Misonix</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

E.의 ε 소단위 대장균 DNA 중합 효소 III는 243 개의 아미노산으로 구성되어 있으며, 잔류 187-243 (17)을 삭제하지 않는 한, 용성 (17, 18)을 갖추고 있습니다. 그러나, 우리는 이전에 전체 길이 α, ε, 및 θ 서브 유니트의 공동 발현이 수용성 DNA 폴리머 라제를 촉매 III 코어 (도 1)을 수득 11 것을 보여 주었다. ⅰ) α와 ε 소단위의 과발현은 독립적 아라비 노...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

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 ...

the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

Research

JoVE Journal

Biology

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

11.8K 조회수.  University of Bologna. 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. 

비디오: The Multifaceted Benefits of Protein Co-expression 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 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 ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

에 박테리아 기능 네트워크 및 경로를 매핑<em&gt; 대장균</em&gt; 합성 유전자 배열을 사용하여

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

에서 펩타이드 및 단백질과 재조합 엘라스틴 같은 폴리 펩타이드 및 그 융해의 비 크로마토 그래피 정제<em&gt; 대장균</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

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 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

갭 접합부 세포 간 통신 : 천연 제품의 독성 물질과 독소의 반작용을 평가하기위한 기능성 바이오 마커, 보건 혜택

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.