저자의 실험실에서 작업은 도이치 포르스충스게마인샤프트 (http://www.dfg.de 의해 지원되었다; CO 1139/1-1), 퐁더 케미셴 인더스트리(http://www.vci.de/fonds), 분자 생물학 괴팅겐 센터(GZMB). 저자는 유용한 의견과 원고의 비판적 독서에 대한 요르그 스튀르케를 인정하고 싶습니다.

1. 한천 접시, 문화 매체, 극저온, 프리컬쳐 준비
<ol><li>성장 매체와 필요한 시약을 준비합니다(재료 및 시약 표 참조).</li>
	<li>B. 서브틸리스 균주(예:. BP40(rocG+ gudBCR amyE::PgudB-yfp)및 BP52(rocG+ gudB+ amyE::PgudB-cfp)각각 1개와 2개의 활성 GDHs를 발현)2는 SP 중간 식도 플레이트에 대한 경쟁 실험에서 단일 콜로니를 얻을 수 있다. 플레이트를 37°C에서 하룻밤 동안 배양합니다.</li>
	<li>단일 콜로니를 가지고 4 ml LB 액체 매체를 포함하는 무균 배양 튜브를 접종하십시오. 28°C와 220 rpm에서 하룻밤 사이에 박테리아를 성장시다.</li>
	<li>세포가 lyse 또는 산발적으로 피하기 위해 28 °C에서 하룻밤 배양을 성장시다.</li>
	<li>표준 분광계를 사용하여 600nm(OD600)의파장에서 배양물의 광학 밀도를 측정합니다.</li>
	<li>배양이 2.0의 OD600에 도달하면 배양의 0.75 ml를 멸균 50% 글리세롤 용액과 혼합하여 1.5ml 반응 튜브에 섞는다. 최종 OD600은 약 108 세포 /ml를 포함하는 냉동 주를 얻기 위해 1.0이어야한다.</li>
	<li>튜브를 -80°C에 저장합니다.</li>
	<li>형광소 유전자를 인코딩하는 cfp 및 yfp로 표지된 각 균주의 3가지 사전 배양을 만듭니다. -80°C 극저온스톡으로부터 1μl 세포로 프리컬쳐(4ml LB 액상 배지를 함유한 멸균 배양 튜브)를 접종한다. 28°C와 220 rpm에서 하룻밤 동안 배양배합니다.</li>
</ol>2. 박테리아의 공동, 샘플 수집 및 샘플 저장
<ol><li>갓 100ml C-Glc 및 CE-Glc 최소 배지(시약 및 재료 표 참조)를 준비하고 각 배지의 20ml를 멸균 100ml 쉐이크 플라스크로 옮기습니다.</li>
	<li>하룻밤 사이에 재배된 배양 전 배양물의 0.1 ml를 1.5ml 의 큐벳에 0.9 ml LB 배지로 희석하고 OD600을결정한다.</li>
	<li>경쟁 실험을 위해, 1.0-1.5 사이의 유사한 OD600을 가진 다른 균주의 그 사전 문화를 취하십시오.</li>
	<li>혼합 세포 집단을 얻기 위해, 적절한 OD600을 20ml C-Glc 및 CE-Glc 최소 배지에서 0.05의 OD 600으로 적절한 OD600을 가진 프리컬쳐의 세포를 100ml 쉐이크 플라스크로 보충하였다. 경쟁 실험을 위해 두 균주는 1:1 비율로 혼합되어야합니다.</li>
	<li>플라스크에서 10ml 샘플을 가져 와서 15 ml 플라스틱 튜브로 옮기고 표준 테이블 상단 원심 분리기에서 4,000 x g에서 10 분 동안 원심 분리로 세포를 수확하고 상퍼를 폐기하십시오.</li>
	<li>0.5 ml 신선한 LB 배지에서 세포를 다시 중단하고 세포를 멸균 1.5 ml 반응 컵으로 옮기습니다. 멸균 글리세롤 50% 0.5 ml를 넣고, 엄격한 소용돌이에 의해 현탁액을 혼합하고, 추가 처리될 때까지 -80°C 냉동고에 샘플을 보관한다.</li>
	<li>37°C 및 220 rpm에서 최대 24시간 동안 쉐이크 플라스크에 남아 있는 세포를 배양한다. 형광의 사진 표백을 방지하기 위해 어둠 속에서 쉐이크 플라스크를 유지합니다.</li>
	<li>7 시간 및 24 시간 성장 후 0.1 ml의 추가 샘플을 가져 가라. 샘플의 1:10 희석(C-Glc 또는 CE-Glc 최소 배지)의 OD600을 측정하고 유의하십시오.</li>
	<li>각 배양에서 적정량의 세포를 섭취하여 1.0의 OD600을 갖는 극저온을 만듭니다. 추가 처리될 때까지 샘플을 -80°C에 저장합니다.</li>
</ol>3. 정량 분석을 위한 샘플 처리, 도금 및 인큐베이션
<ol><li>모든 샘플을 수집한 후, 냉동주를 해동하고, 세포를 0.9% 식염수 용액(시약 및 재료 표 참조)에서 최대10-3으로희석한다.</li>
	<li>SP 중간 식기 판에10-3 희석의 플레이트 0.1 ml를 멸균 유리 파이펫을 사용하여 세포를 분배한다.</li>
	<li>단일 식민지가 나타나기 전까지 어둠 속에서 밤새 플레이트를 37 °C에서 배양하십시오.</li>
</ol>4. 정량 분석을 위한 스테레오 형광 현미경 검사법에 의한 생존자 계산
<ol><li>스테레오 형광 현미경의 생존자를 계산하는 동안 더 나은 방향을 위해 네 부분으로 검은 펜으로 한천 판의 바닥을 분할합니다.</li>
	<li>접시를 현미경 아래에 거꾸로 놓고 콜드 라이트 소스를 사용하여 식민지를 초점으로 가져옵니다.</li>
	<li>콜로니가 초점을 맞추고 나면 CFP에 대한 적절한 필터 세트로 전환하여 cfp-labeled균주의 생존 셀을 시각화합니다. 이 균주의 생존자를 펜으로 표지하여 계산하고 숫자를 기록합니다.</li>
	<li>에탄올로 라벨을 제거하고 YFP필터 세트로 전환하여 yfp-labeled 스트레인의 생존 셀을 시각화합니다. 콜론에 펜으로 라벨을 붙여 생존자를 다시 계산하고 숫자를 기록합니다.</li>
</ol>5. 반정량 분석을 위한 샘플 처리 및 현미경 검사법
<ol><li>예시적인 수치의 경우, SP 한천 판에서 3.1단계에서10-4 희석(약 100셀)의 10μl을 반점한다(그림 3참조).</li>
	<li>단일 식민지가 나타나기 전까지 어둠 속에서 밤새 플레이트를 37 °C에서 배양하십시오.</li>
	<li>페트리 접시를 현미경 아래에 뚜껑없이 놓고 차가운 광원을 사용하여 그 자리를 집중시하십시오.</li>
	<li>일러스트레이션 인물을 위해 반점 사진을 찍어보세요. 차가운 광원으로 식민지의 사진을 찍기위한 적절한 노출 시간을 선택하십시오.</li>
	<li>플레이트를 이동하지 않고 CFP 필터 세트로 변경하고 노출 시간을 조정하고 사진을 찍습니다. YFP 필터 세트와 동일한 작업을 수행하 고 추가 분석을 위해 사진을 저장 합니다.</li>
</ol>6. 데이터 분석
<ol><li>정량적 데이터 분석에 Excel과 같은 소프트웨어를 사용합니다. 계산된 식민지에 따라, 100%로 설정된 식민지의 전체 수에 대하여 노란색과 파란색 식민지의 백분율을 계산합니다.</li>
	<li>계산된 숫자를 사용하여 누적된 막대 다이어그램을 만듭니다(그림 4 및 그림 5참조). Adobe Photoshop과 같은 이미지 처리 프로그램을 사용하여 다른 지점에서 찍은 사진의 병합된 사진을 생성합니다. 또는 <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> 다운로드가능한 자유롭게 사용할 수 있는 소프트웨어 ImageJ를 이미지 프로세싱에 사용할 수 있습니다.</li>
	<li>CFP 필터 세트와 YFP 필터 세트로 촬영한 한 지점에서 사진을 엽니다. 콘트라스트와 밝기를 최적화하여 미디어의 배경 형광을 줄입니다.</li>
	<li>사진 중 하나로 이동하여 모든 것을 선택합니다. 그림을 복사하여 다른 그림에 붙여 넣습니다.</li>
	<li>함수 "색상 회피"를 사용하여 사진을 병합하거나 채널 탭을 사용하여 형광 사진을 오버레이합니다. 다른 매체와 다른 시간 지점에서 식민지의 병합 된 사진은 액체 문화 내에서 다른 균주의 성장을 나타냅니다.</li>
</ol>7. 특정 팁: 염료 스위치 실험 및 cfp 및 yfp로 표시 된 동종 균주의 공동
B. 서브틸리스에 있는 2개의 불소-인코딩 유전자의 표정은 적합그리고 따라서 박테리아의 성장 속도에 영향을 미칠 수 있습니다. 따라서, 재배 중 세포 집단으로부터 하나의 경쟁자 균주를 제거하는 것은 단순히 플루오로포어의 부정적인 효과로 인한 것을 배제하기 위해 다음과 같은 실험을 수행하는 것이 좋습니다.
<ol><li>전체 실험을 반복하고 반비례표시균(예를들어, 이전 cfp-표지된균주는 이제 yfp 유전자로 표지되고 그 반대의 경우도마찬가지입니다). 비록 반전, 얻은 결과 한 변형 다른 변형에 비해 선택적 성장 이점을 가지고 이전 관측에 필적 해야.</li>
	<li>yfp 및 cfp(예를 들어 BP40)와 cfp(예를 들어, BP40+gudB CR amyE::PgudB-yfp)및 BP41(rocG+ gudBCR amyE::PgudB-cfp)로표시된 전체 실험 및 공동 재배 이소생균균을 반복한다. 시간이 지남에 따라 세포 집단의 조성에 따라 박테리아의 적합성에 대한 두 형광중 하나의 부정적인 효과를 추정한다.</li>
</ol>

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Biotechnol. 21, 20-35 (2011).</li><li>Rabatinov&#225;, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&#948;+subunit+of+RNA+polymerase+is+required+for+rapid+changes+in+gene+expression+and+competitive+fitness+of+the+cell.">The &#948; subunit of RNA polymerase is required for rapid changes in gene expression and competitive fitness of the cell.</a> J. Bacteriol. 195, 2603-2611 (2013).</li><li>Capra, E. J., Perchuk, B. S., Skerker, J. M., Laub, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+mutations+that+prevent+crosstalk+enable+the+expansion+of+paralogous+signalling+protein+families.">Adaptive mutations that prevent crosstalk enable the expansion of paralogous signalling protein families.</a> Cell. 150, 222-232 (2012).</li><li>Garc&#237;a-Betancur, J. C., Yepes, A., Schneider, J., Lopez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+Bacillus+subtilis+biofilms+using+fluorescence+microscopy+and+flow+cytometry.">Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry.</a> J. Vis. Exp. (15), (2012).</li></ol>

저자는 경쟁적인 재정적 이익이 없다고 선언합니다.

박테리아16의경쟁력있는 적합성을 분석하기 위해 몇 가지 방법이 개발되었습니다. 많은 경우에 박테리아는 다른 항생 저항 카세트17로표지되었습니다. 우리의 접근 방식과 유사하게, 항생제 저항 카세트를 가진 세포의 라벨링은 정의된 성장 조건하에서 박테리아의 경쟁적 적합성의 평가를 허용합니다. 더욱이, 이 방법은염색체(17)에특정 궤적에서 서로 다른 세포의 경쟁?...

토양 박테리아는 일반적으로 유연한 규제 네트워크와 광범위한 신진 대사 용량을 부여합니다. 두 가지 기능을 모두 사용하면 세포가 쌍백과 단백 동화 경로를 조정하여 주어진 생태 틈새 시장1에서사용할 수있는 영양소를 위해 동료 및 기타 미생물과 경쟁 할 수 있습니다. 그러나, 박테리아가 그들의 환경에 적응할 수 없는 경우에 그밖 기계장치는 종의 생존을 설명할 수 있습니다. 실제로, 많은 박테리아가 빠르게 증식하고 인구가 높은 세포 밀도에 도달할 수 있기 때문에 인구 하위 인구는 선택적 성장 이점을 가진 세포를 제공하고 그러므로 그들의 적정성을 증가시키는 유익한 돌연변이를 자발적으로 축적할 수 있습니다. 더욱이, 돌연변이 핫스팟 및 스트레스 유발 적응 돌연변이 발생은 부적응된 박테리아2,3의진화를 용이하게 할 수 있다. 따라서, 연속선택 하에서 돌연변이와 성장의 축적은 동일한4,5세내에서도 엄청난 미생물 다양성의 기원이다. 자연에서와 마찬가지로, 세균 게놈의 형성은 또한 선택하에 지속적인 재배 때문에 실험실에서 발생합니다. 이것은 기본 연구 및 산업에서 전 세계적으로 사용되는 그람 양성 박테리아 B. subtilis의 길들여지는 것으로 예시됩니다. 1940년대에 B. 서브틸리스는 DNA 손상 엑스레이로 처리되었고, 그 다음에는 특정 성장 조건 하에서 재배하였다6. 이들의 가축화 과정에서 축적된 돌연변이는 많은 성장 특성의 상실을 차지하고, 즉 B. 서브틸리스 실험실 균주(168)는 복잡한 식민지를 형성하는 능력을상실하였다 7,8. 
요즘, 가장 잘 연구 된 모델 박테리아 Escherichia 대장균과 B. subtilis에대 한, 다양 한 강력한 도구는 유전 특정 과학적 질문을 해결 하기 위해 그들의 게놈을 조작하는 사용할 수 있습니다. 때때로 관심 유전자의 불활성화는 심각한 성장 결함을 일으키는 원인이 되고, 그 때 표준 성장 매체9에서명확하게 보입니다. 대조적으로, 약한 성장 결함을 일으키는 원인이 되고 이렇게 약간 만 긴장의 적각에 약간 영향을 미치는 돌연변이는 수시로 무시됩니다. 그러나, 두 경우 모두 여러 세대에 대한 돌연변이 균주의 장기간 인큐베이션 및 패시징은 일반적으로 부모 균주2,9의표현형을 복원한 억제제 돌연변이체의 축적을 초래한다. 억제제 돌연변이의 특성화 및 부모 돌연변이 균주의 성장 결함을 복원한 돌연변이의 식별은 중요하고 종종 새로운 세포 과정의 명분보정을 허용하는 매우 유용한접근법이다 10,11.
우리는 B. subtilis12에서글루타민성 항상성의 제어에 관심이 있습니다. 대장균과유사하게, B. 서브틸리스는 억제제 돌연변이의 축적에 의해 글루타민트 항상성(즉, 글루타민제 분해2의블록)의 변투에 반응한다. 자발적인 돌연변이에 의해 획득한 이들 억제돌연변이의 게놈 변화는 글루타민트 항상성을 급속히 회복시키는 것으로 나타났다9,13. 따라서, 박테리아의 국산 후 특정 성장 상태에 B. 아틸리스의 적응이 효소 합성및 조미료 대사에 관여하는 진화된 효소 활동에서 미러링된다는 것은 놀라운 일이아니다.12. 국내화 과정에서 성장 배지에서 외인성 글루타민의 부재가 실험실 균주 1682,14에서비밀글루타민제 탈수소효소(GDH) gudBCR 유전자의 출현 및 고정을 위한 원동력이 되었다는 것이 시사되고 있다. 이러한 가설은 실험실 균주에서 의한 GDH 활성의 감소된 양이 외인성 글루타민산염이 부족할 때 선택적 성장 이점을 박테리아에 제공한다는 우리의 관측에 의해 지원된다2. 더욱이, B. subtilis 균주의 재배, GDH GudB를 합성, 외인성 글루타민의 부재시 gudB 유전자2를불활성화한 억제제 돌연변이의 축적을 초래한다. 명백하게, 이화 활성 GDH의 존재는 비물질에 사용될 수 있는 내인성 생산 글루타민산염이 암모늄및 2-oxoglutaraterate(도1)로저하되기 때문에 세포에 불리하다. 대조적으로, 글루타민이 매체에 의해 제공될 때, 높은 수준의 GDH 활성을 갖춘 B. 서브틸리스 균주는 하나의 기능성 GDH만 합성하는 균주에 비해 선택적 성장 우위를 가지게 한다. 높은 수준의 GDH 활성이 박테리아가 중간2에서 제공하는 다른 탄소 공급원 이외에 글루타민을 제2 탄소 원으로 활용할 수 있다고 가정하는 것이 합리적이다(도 1참조). 따라서, GDH 활동은 외인성 글루타민산염의 가용성에 따라 박테리아의 적합성에 강하게 영향을 미친다.
여기서 우리는 염색체에 단일 궤적(도 2)에서다른 두 B. 아틸리스 균주 사이의 종 내 경쟁을 모니터링하고 시각화하는 매우 예시적인 방법을 제시한다. 두 균주는 형광YFP와 CFP를 인코딩yfp 및 cfp 유전자로 표시하고, 다른 영양 조건하에서 cocultivate. 시간이 지남에 따라 샘플링하고 각 배양소의 생존자는 일반적인 스테레오 형광 현미경을 사용하여 쉽게 모니터링 할 수 있습니다. 본 논문에 기재된 절차는 시간이 지남에 따라 세포 집단에서 각각 유익하고 해로운 돌연변이의 급속한 클로날 팽창 및 제거를 시각화하기 쉽고 적합합니다.

<table><tbody><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>3746</td><td></td></tr><tr><td>Agar</td><td>Difco, USA</td><td>214010</td><td></td></tr><tr><td>Ammonium ferric citrate (CAF)</td><td>Sigma-Aldrich, Germany</td><td>9714</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&amp;nbsp;&lt;/sub&gt;</td><td>Roth, Germany</td><td>5239</td><td></td></tr><tr><td>Glucose</td><td>Applichem, Germany</td><td>A3617</td><td></td></tr><tr><td>Glycerol</td><td>Roth, Germany</td><td>4043</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth, Germany</td><td>6878</td><td></td></tr><tr><td>KCl</td><td>Applichem, Germany</td><td>A3582</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth, Germany</td><td>3904</td><td></td></tr><tr><td>KOH</td><td>Roth, Germany</td><td>6751</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Roth, Germany</td><td>P027</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>T881</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck Millipore, Germany</td><td>102786</td><td></td></tr><tr><td>NaCl</td><td>Roth, Germany</td><td>9265</td><td></td></tr><tr><td>Nutrient broth</td><td>Roth, Germany</td><td>X929</td><td></td></tr><tr><td>Potassium glutamate</td><td>Applichem, Germany</td><td>A3712</td><td></td></tr><tr><td>Tryptone</td><td>Roth, Germany</td><td>8952</td><td></td></tr><tr><td>Tryptophan</td><td>Applichem, Germany</td><td>A3445</td><td></td></tr><tr><td>Yeast extract</td><td>Roth, Germany</td><td>2363</td><td></td></tr><tr><td>1.5 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,690,001</td><td></td></tr><tr><td>2.0 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,691</td><td></td></tr><tr><td>15 ml Plastic tubes with screw cap</td><td>Sarstedt, Germany</td><td>62,554,001</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt, Germany</td><td>82.1473</td><td></td></tr><tr><td>1.5 ml Polystyrene cuvettes</td><td>Sarstedt, Germany</td><td>67,742</td><td></td></tr><tr><td>15 ml Glass culture tubes&amp;nbsp;</td><td>Brand, Germany</td><td>7790 22</td><td></td></tr><tr><td>100 ml Shake flasks with aluminium caps</td><td>Brand, Germany</td><td>928 24</td><td></td></tr><tr><td>Sterile 10 ml glass pipettes</td><td>Brand, Germany</td><td>278 23</td><td></td></tr><tr><td>Incubator (28 and 37 &amp;deg;C)</td><td>New Brunswick</td><td>M1282-0012</td><td></td></tr><tr><td>Standard pipette set (2-20 &amp;mu;l, 10-100 &amp;mu;l, 100-1,000 &amp;mu;l)</td><td>Eppendorf, Germany</td><td>4910 000.034, 4910 000.042,</td><td></td></tr><tr><td>Table top centrifuge for 1.5 and 2 ml reaction tubes</td><td>Thermo Scientific, Heraeus Fresco 21, Germany</td><td>75002425</td><td></td></tr><tr><td>Table top centrifuge for 15 ml plastic tubes</td><td>Heraeus Biofuge Primo R, Germany</td><td>75005440</td><td></td></tr><tr><td>Standard spectrophotometer</td><td>Amersham Biosciences Ultrospec 2100 pro, Germany</td><td>80-2112-21</td><td></td></tr><tr><td>Stereofluorescence microscope&amp;nbsp;</td><td>Zeiss SteREO Lumar V12, Germany</td><td>495008-0009-000</td><td></td></tr><tr><td>Freezer (-20 and -80 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Fridge (4 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Autoclave</td><td>Zirbus, LTA 2x3x4, Germany</td><td>-</td><td></td></tr><tr><td>pH meter</td><td>pH-meter 766, Calimatic, Knick, Germany</td><td>766</td><td></td></tr><tr><td>Vortex</td><td>Vortex 3, IKA, Germany</td><td>3340000</td><td></td></tr><tr><td>Balance</td><td>CP2202S, Sartorius, Germany</td><td>replaced by</td><td></td></tr><tr><td>Black pen (permanent marker)</td><td>Staedler, Germany</td><td>317-9</td><td></td></tr><tr><td>Powerpoint program</td><td>Microsoft, USA</td><td>-</td><td></td></tr><tr><td>Office Excel program</td><td>Microsoft, USA</td><td>-</td><td>Program for data processing</td></tr><tr><td>Adobe Photoshop CS5</td><td>Adobe, USA</td><td>replaced by CS6, download</td><td>Computer program for image processing</td></tr><tr><td>Computer</td><td>PC or Mac</td><td>-</td><td></td></tr><tr><td>ZEN pro 2011 software for the stereofluorescence microscope</td><td>Zeiss, Germany</td><td>410135 1002 110</td><td>AxioCam MRc Rev. Obtained through Zeiss</td></tr><tr><td colspan="4">&lt;strong&gt;Specific solution recipes&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;SP Medium&lt;/strong&gt;</td></tr><tr><td>8 g Nutrient broth</td><td></td><td></td><td></td></tr><tr><td>0.25 mg MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>1 g KCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid SP medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>1 ml CaCl&lt;sub&gt;2&lt;/sub&gt; (0.5 M), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>1 ml MnCl&lt;sub&gt;2&lt;/sub&gt; (10 mM), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;LB Medium&lt;/strong&gt;</td></tr><tr><td>10 g Tryptone</td><td></td><td></td><td></td></tr><tr><td>5 g Yeast extract</td><td></td><td></td><td></td></tr><tr><td>10 g NaCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid LB medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;C-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%)</td><td></td><td></td><td>autoclaved for 20 min at 121 &amp;deg;C</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;CE-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>20 ml Glutamate (40%)</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%), autoclaved for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>5 x C salts&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>20 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>80 g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>16.5 g (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>0.232 g MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>12.3 g MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O, autoclave for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>40% Glutamate solution</td><td></td><td></td><td></td></tr><tr><td>200 g L-Glutamic acid</td><td></td><td></td><td></td></tr><tr><td>adjust the pH to 7.0 by adding approximately 80 g KOH</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;0.9% Saline (NaCl) Solution&lt;/strong&gt;</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>50% Glycerol solution</td><td></td><td></td><td></td></tr><tr><td>295 ml Glycerol (87%)</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">Bacteria (All strains are based on the &lt;em&gt;Bacillus subtilis&lt;/em&gt; strain 168)</td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP40 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-yfp)&amp;nbsp;</td><td>Laboratory strain collection</td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP41 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-cfp)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP52 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-cfp)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP156 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-yfp)</td><td></td><td></td><td></td></tr></tbody></table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

박테리아와 같은 많은 미생물은 매우 빠르게 증식하고 인구는 높은 세포 밀도에 도달 할 수 있습니다. 인구에 있는 세포의 작은 분수는 항상 세포에 해를 끼치거나 유익한 돌연변이를 축적했습니다. 돌연변이의 체력 효과가 강한 선택적 성장 이점을 가진 하위 인구를 제공하는 경우에, 이 하위 인구의 개별은 급속하게 경쟁하고 그들의 즉각적인 동료를 완전히 제거할 수 있습니다. 따라서, 유익한 돌연변이를 획득한 세포의 작은 유전적 변화 및 선택 중심축적은 세포 집단의 유전자형의 완전한 변화로 이어질 수 있다. 여기서 우리는 그람 양성 모델 박테리아 부틸리스의형광 표시 개인의 공동 재배에 의해 시간이 지남에 따라 세균 세포 인구에서 각각 유익하고 해로운 돌연변이의 급속한 클로날 팽창 및 제거를 모니터링하는 절차를 제시한다. 이 방법은 세균 세포 집단의 개인 들 간의 종 내 경쟁을 표시하는 것이 쉽고 매우 예시적입니다.

여기서 설명된 방법은 각각 형광경 CFP와 YFP를 인코딩하는 cfp 및 yfp 유전자로 표지된 B. 서브틸리스 균주로 구성된 세포 집단에서 종 내 경쟁을 시각화하기 위해 성공적으로 적용되었다. 도 3에도시된 바와 같이, 이 방법은 매우 예시적인 방식으로 종내 경쟁을 시각화하는 데 사용될 수 있다. 작은 부위에 샘플을 발견함으로써 세포 집단의 복제 구성을 한눈에 볼 수 ...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

박테리아는 그들의 일생 도중 해롭거나 유익한 돌연변이를 축적할 수 있습니다. 유익한 돌연변이를 축적한 세포 개인의 인구에서 그들의 동료를 급속하게 능가할 수 있습니다. 여기서 우리는 형광으로 표시된 개별을 사용하여 시간이 지남에 따라 세균성 세포 인구에 있는 종 경쟁을 구상하는 간단한 절차를 제시합니다.

형광표균의 세균세포 인구에 대한 종 내 경쟁 모니터링

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.5K 조회수.  Georg-August University. 박테리아는 그들의 일생 도중 해롭거나 유익한 돌연변이를 축적할 수 있습니다. 유익한 돌연변이를 축적한 세포 개인의 인구에서 그들의 동료를 급속하게 능가할 수 있습니다. 여기서 우리는 형광으로 표시된 개별을 사용하여 시간이 지남에 따라 세균성 세포 인구에 있는 종 경쟁을 구상하는 간단한 절차를 제시합니다.

비디오: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

표면 식민지 미생물 집단에 공간 분리를 모니터링

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

연구

유전학

Title Cell Encapsulation by Droplets

My name is Hang Moon. I'm working with the professor of condoms. He and me work with the XYJ stage to make a 3D structure of cell.Hi, I am PE and Lynn and I'm working with the cell printing project with cell encapsulation and media and gels. This is solenoid be for ejection of a cell and cell mixture of aros over cell media. The so valve is activated by two signal line.The signal line is came from a dry board here. Signal came from here. That line through the that line, that one is the function first generator.So first is generated by our programmed way. So if you want, you want to change the purse, you can change the program with the that switches line or you can control that process generator to computer. The cellular valve is open and closed by the electric signal, but the, the cell is supplied through that line.The cell and the media or agro gel is supplied through the syringe syringes. The syringes inside of the water, inside of syringe is the press light by the air. The pressurized air is supplied from a tank like here the line.So the little line and the ized air is fly through the air filter. The air filter is discriminate the dust and other kind of the dirt. That is pressure generator we call it, is generating the signal used for open the solar air bell or closed the solar air valve computer is the connected through P-I-B-U-S-V connector.So we can use the USB port by directly our computer, the laptop computer. So that one is connected to that side here. And then we can control through the control laptop here.So if we want to synchronize of the movement of the stage and opening the bell is can be controlled by the same controller as a laptop. So we can control the timing when it open, when it close, and when it moves and when it stops. And we can synchronize the motion and opening bell.That stage is the motorized stage. It is automatically automatically moved by through the death signal line and motor. So motorization is 0.1 micrometers.So it can ideally it can draw the line up to 100 millimeter line. So jet axis is moving, going up and down like that way. So it control the cap between the substrate and the solar belt.So it can control the height of ejection. That substrate is the connected to the XY stages here, X here and Y there. So it's moving around a plan XY plan.So it draws some feature demo right stages is three Xs, X, Y, and G.So the jet con jet axis or XY axis is controlled by signal here. So signal line is connected to the motion controller here. That motion controller doesn't have any manual switch at all because it cannot be controlled manually.It's controlled through the laptop computer. So laptop computer is connected. The backside of the controller, we have three xs for a printing cell XY jet.So it make three different kind of signal independently. And then motion controller is communicate through the rf 2, 3, 2 connectors is directly connected to the USV. And then USV is connected to laptop computer.So laptop controlled borrower. We use the AutoCAD program, we make a gen A path like that way bam. And then it change to the machine language like that.So machine language is transferred to the like that the simple character and numbers like that. There is each position related to the X, Y, Z stages here I choose the file, contain that kind of program and then open downloading program like that way. And then I choose from file like does exact the same, the text file.Just the text file is downloaded, choose text file and automatically download from laptop to com motion controller executive and then it going the BAM character as I mentioned before, BAM. In this flask are N IH 3G three mouse fiberblast cells. And I'm going to first aspirate the old media.This process is to passage the cells and the cells are adhered to the bottom of the flask. So after aspirating the media, I'm going to put in six RYS in which is an enzyme.Okay? So after the trypsin has been put into the flask, we need to wait three to five minutes for the cells to become detached from the bottom of the flask.Now we're ready to put in the media and passage the cells into a centrifuge tube. It's a one-to-one dilution. So I'm putting in six mil of this media right here and I'm gonna wash out the saws a little bit to make sure all of them are passaged.And now the cells are ready to be centrifuge in this tube. Now we're going to centrifuge these cells at a thousand RPM three to five minutes. Now that the cells have been spun down into a pellet, I'm going to aspirate the supernatant.Now I'm going to wash out the cells with PBS. And this is to, this is for staining the cells. The stains that we are using are esterase substrates.Okay, now we we're going to centrifuge again and spin down the cells into a palate. And I'm going to aspirate the PBS and I'm going to resin the cells. I'm in media to be counted.I'm mixing the cells so that when we take the a sample of it, the concentration will be even throughout the tube. Now I'm going to take a hundred microliters of the cells to measure the cell density. Now I'm putting in a hundred microliters of type and blue, which is a stain.We're going to wait three to five minutes in order for the stain to permeate dead cell membranes. When we count the cells, the blue ones we don't count. The cells are ready to be counted with the hemo cytometer, I'm going to transfer 50 microliters of the cell mixture to each side of the hemo cytometer.Okay, I'm going to cover it with a glass slide side. There's two sides to the hemo cytometer. I'm gonna count the first side and after counting both sides, I'm going to take the average and then multiply by two because we had a one-to-one dilution with the type in blue.So basically our cell density will be 135 times 10 to the four cells per mil. For our experiment, we will be using a cell solution with a concentration of half a million cells per mil. And so since we have 13.5 million cells, I will be resus suspending the cells in 27 mil of media.We are using two different types of stains. This is a live dead assay from molecular probes. Our first stain is called calcium am.Our second stain is called AUM homodimer. It stains dead cells. We use blue excitation to get green fluorescence and green excitation to get red fluorescence For these two stains, when we're imaging our dice solution will be prepared in PBS.So first I'm going to transfer five mil of PBS into a tube phosphate buffered saline. That's a saline solution. Now I'm going to add half a microliter of calcium am per mil.Now I'm adding 2.5 microliters of calcium am to the tube and two microliters of athe homodimer per mil of PBS. Here is the btex machine. It makes the cell and media well distributed through the whole volume.Now we load the cell 200 microliter onto the seven here. And then we put on the cap, this is a tight tightly sealed through like that way here I open the valve and then it's the pressure light air comes in and then I adjust the belt like that. And then it change the pressure.So I adjust. The pressure is five PSI. The inside of tubing pressure is five PSI.From here to here, it connected through the this inside the hole. So just we open the bell here and then the cell injected with the five PSI pressure and then starting open. And then Sona valve starting operate.And then we roll the substrate. It substrate is just a simple slide glass. And then we load the proper portion and I put some sketch tape to fix the substrate like that, right?And simply I just fix it. So we are setting whole thing the cell and ary valve and the substrate during the moving the ary working, start working. So we are set up whole thing and then open ary valve start.And then we move the stage like that, right? And then we make some pattern onto the substrate pattern onto the substrate and bam character. I'm going to immerse these droplets that we printed in this dye solution that we made earlier.And now the droplets will be incubated for 10 minutes and then imaged. First I'm going to adjust the image through the eyepiece. Our first image is just a bright field image.I'm gonna turn off the light and change the filter so that we have blue excitation, which will show green fluorescence. And the third image we'll be taking is the dead cells, which is the Thm Homodimer stain. And with green light, we will get red fluorescence.

흩뿌려진의 제목 셀 캡슐화

생물학

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS로 인한 세균 경쟁을 모니터링 할 비주얼 분석

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

면역학 및 감염병학

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

유전자 변형으로 인코딩된 칼슘 센서를 사용하여 Apoptosis 동안 Mitochondrial 칼슘 레벨에서 동적 변경 사항 모니터링

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

비브리오 피셔리 분리 사이의 경쟁 적 상호 작용을 정량화하기위한 동전 분석

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

단세포 종 간 세균성 상호 작용의 운동 시각화

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

화학적 매개 종간의 상호 작용을 검출하는 공 배양을 사용하여

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

단세포 형광 화상 진찰을 사용하여 세균 간 경쟁의 정량화

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://app.jove.com/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.