유전체학 및 진화 과학을 전진 파지 사냥꾼 (SEA-파지) 프로그램 - 저자는 액티브 교육에 대한 게놈 컨소시엄 (GCAT)와 HHMI - 과학 교육 얼라이언스와 말콤 캠벨 토드 Eckdahl을 인정하고 싶습니다. 이 프로젝트는 연구 자원을위한 국립 센터 (P20RR016460)와 국립 보건원에서 일반 의료 과학 국립 연구소 (P20GM103429)에서 보조금에 의해 지원되었다. 이 연구는 부여 번호의 IIA-1457888 아래에있는 국립 과학 재단 (National Science Foundation)에 의해 부분적으로 지원되었다. 또한 기관 (우아 치타 침례 대학) 기금은 JD 패터슨 여름 연구 활동을 통해 제공되었다.

1. 설계와 적절한 스티커 엔드와 주문 올리고 뉴클레오티드 <ol><li> 자유롭게 사용할 수 온라인 프로그램을 사용하여 게놈 분석을 통해 잠재적 인 RHO 독립 종결을 확인합니다. (7) </li><li> 이중 가닥 DNA로 작업 할 때, 터미네이터의 방향 테스트 할 결정합니다. 7 PGR - 블루 플라스미드는 상단 (정 가닥)의 방향 &#39;3&#39;이 5 결찰 터미네이터를 확인합니다. <ol><li> , 무료 온라인 소프트웨어를 사용하여 PGR - 블루에서 테스트를 위해 원숭이를 예를 순서 방향을 재 그 반대로 보수로 바닥 (역) 가닥 터미네이터를 변환합니다. </li></ol></li><li> 방향이 정확한 것으로 결정되면, 상부 가닥 &#39;말단 (5)에 접착 단부 &quot;5&#39;- CGAC-3&#39; &#39;를 추가한다. . 바닥 가닥의 끝 &#39;이 5 끈끈한 끝 &quot;5&#39;-CCGC-3&quot;&#39;추가 (8)이 그 결찰 삽입 적절한 삽입됩니다 보장합니다방향 GGA를 사용하여. </li><li> 서열이 결정되면, 상업적으로 단일 가닥 올리고 뉴클레오티드로 주문한다. </li></ol> 2. 어닐링 올리고 뉴클레오티드는 (동결 건조하는 DNA를 만 적용) <ol><li> 100 μM의 농도로 클레아 유리수 개별 올리고 다시 중단. </li><li> 1 M의 NaCl 100 mM 트리스 - 염산 pH가 7.4 : 10 배 어닐링 버퍼를 확인합니다. 어닐링 버퍼 몇 주 동안 4 ℃에서 저장 될 수있다. 어닐링 버퍼로 이루어지고, 올리고 뉴클레오티드는 다시 중단되면, 어닐링 8 시작한다. </li><li> 각 테스트가 종료 될 때까지, 1.5 ml의 마이크로 원심 튜브에 초순수의 16 μL를 추가한다. </li><li> 각 microcentrifuge 관에 10 배 어닐링 버퍼 2 μl를 추가합니다. </li><li> 원하는 터미네이터에서 상단 가닥 올리고 뉴클레오티드의 1 μL (100 μM)를 추가합니다. </li><li> 원하는 터미네이터에서 바닥 가닥 올리고 뉴클레오티드의 1 μL (100 μM)를 추가합니다. </li><li> 끓는 물 바 만들기핫 플레이트 수돗물 장소 약 400ml를 함유하는 1000 ml의 비이커를 사용 번째. </li><li> 부동의 단계 2.6에서의 microcentrifuge 튜브를 넣고 4 분간 끓인다. 4 분 후, 열판을 해제하지만, 튜브를 남겨 수조 천천히 차가운 O / N (18 시간) 8 로트. -20 ° C에서 열처리 터미네이터를 저장합니다. </li></ol> 3. 골든 게이트 총회 (표준 믹스 - 비용 효율적인) <ol><li> 어닐링 터미네이터의 40 nM의 희석을 확인합니다. 어닐링 터미네이터 믹스 (100 μM) 1 μL에 뉴 클레아없는 물 124 μl를 추가합니다. <ol><li> 얼음에 30 만 XG에 초 및 저장을위한 원심 분리기 샘플. </li></ol></li><li> 열 순환기에 대한 새로운 튜브의 적절한 사용되는로, 클레아없는 물 6 μl를 추가합니다. </li><li> 1 상업 DNA 리가 버퍼의 μL [300 mM 트리스 - 염산 (pH를 7.7-7.8), 100 밀리미터의 MgCl 2, 100 mM의 DTT, 10 mM의 ATP]를 추가합니다. 얼음에 모든 후속 단계를 수행합니다. </li><li> 더하다PGR - 블루 (35-50 NG / μL)에 microcentrifuge 관에 대상 플라스미드의 1 μL. </li><li> microcentrifuge 관에 40 nM의 어닐링 터미네이터 1 μl를 추가합니다. </li><li> microcentrifuge 관에 0.5 μL 상업 고성능 BsaI의 제한 효소를 추가합니다. </li><li> microcentrifuge 관에 상업 DNA 리가 아제의 0.5 μl를 추가합니다. </li><li> 120 초 동안 10,000 × g으로 반응 혼합물을 원심 분리기. 그런 다음 열 순환기에 직접 튜브를 배치합니다. </li><li> 37 ° C에서 15 분의 1주기 다음 16 ℃에서 1 분 뒤에 37 ° C에서 1 분 20주기, 상기 열 순환기 프로그램을 실행합니다. 사용 샘플을 즉시 또는 -20 ° C에서 동결 및 저장. (8) </li></ol> 4. 골든 게이트 총회 (상용 마스터 믹스 - 시간 효율적인) <ol><li> 이 40 nM의에 터미네이터 DNA 농도를 희석 2 단계에서 만든 터미네이터를 어​​닐링 100 μM의 1 μL에 뉴 클레아없는 물 124 μl를 추가합니다.새로운에서이 단계를 수행, 1.5 ML의 microcentrifuge 관을 표시. </li><li> 열 순환기에 대한 새로운 튜브 적절한로, 클레아 무료 물을 14 μl를 추가합니다. 새로운 튜브를 따라 표시되어 있는지 확인합니다. </li><li> microcentrifuge 관에 상용 마스터 믹스 버퍼 2 μl를 추가합니다. </li><li> microcentrifuge 관에 PGR - 블루 플라스미드의 1 μL (35-50 NG / μL)를 추가합니다. </li><li> microcentrifuge 관에 40 nM의 어닐링 터미네이터 2 μl를 추가합니다. </li><li> microcentrifuge 관에 상업 마스터 믹스 1 μl를 추가합니다. </li><li> 120 초 동안 10,000 XG에 40 나노 튜브를 원심 분리기. 그런 다음 열 순환기에 직접 튜브를 배치합니다. </li><li> 55 ° C에서 15 분의 1주기 다음 37 ° C에서 60 분의 1주기 다음 열 순환기 프로그램을 실행합니다. 사용 샘플 -20 ° C에서 즉시 또는 동결 및 저장. </li></ol> 5. 변환 및 식민지 선택 <ol><li> 준비 1 ㎜의 정광을 포함하는 페트리 접시 한천 루리아 자료 (LB)암피실린의 이온 (앰프) 및 ​​아라비 노스 (ARB)의 10 mM의 농도. 이 스톡 용액은 이후 단계에서 사용되기 때문에 1 M의 L -arabinose 원액의 적어도 10 ㎖를 준비한다. </li><li> 어떤 높은 효율적인 사용 (8월 10일부터 9월 10일까지 형질 전환 / μg의)를 화학적으로 유능한 E. 형질 전환 대장균 (JM109) 세포. 최상의 결과를 얻으 9 유능한 세포의 50 μL에 연결 반응의 3-5 μl를 추가합니다. 화학적 적격 세포를 사용하는 경우, 세포가 사용되는 특정 변환 프로토콜을 따른다. </li><li> 멸균 L 자형 &quot;하키 스틱&quot;을 사용하여 판 한천 미리 예열 LB (앰프 / ARB)에 형질 전환 세포의 절반 (25 μl를) 확산 및 37 ° C에서 O / N을 품어. 때문에 분해의 암피실린의 빠른 속도로 24 시간 동안 37 ° C에서 부화하지 않습니다. </li><li> 육안 검사에 의해 색상에 따라 식민지 선택을 수행합니다. 성공적인 내고는 / 흰색 가시 광선에 노란색하고 운데 녹색 형광한다R 블루 (450 ㎚) 또는 자외선. 20 시간 - 실패 결찰 (18) 후 가시 광선 아래에서 색 파란색 식민지를 생성합니다. </li></ol> 6. 확인과 터미네이터의 정량화 <ol><li> 1 ㎜의 암피실린 및 아라비 노스 10 mm의 5 ml의 멸균 LB-국물 튜브를 준비합니다. 필요한 튜브의 수는 테스트되는 터미네이터 플러스 컨트롤 (셀 암피실린 내성과 형광 않으며 미가공 PGR 블루 플라스미드를 함유하는 세포)의 수에 의존한다. </li><li> 가시 광선에서 멸균 루프를 사용하여 2-3 흰색 / 노란색 식민지 (단계 5.4)을 선택합니다. 샘플은 37 ° C 및 16 ~ 18 시간 동안 160 rpm으로 진탕에 국물에 품어 할 수 있습니다. 이상 24 시간 동안이 샘플을 품어하지 마십시오. <ol><li> 선택적으로, 적당한 세포의 성장을 보장하기 위해 600 nm의 (OD 600)에서 광 세포 밀도를 결정하는 분광 광도계를 사용한다. 일반적으로, 0.8 ~ 600의 OD 성장의 16 ~ 18 시간 후에 관찰되었다. </li></ol></li><li> 용도나머지 단계에 대한 멸균 96 웰 플레이트. 무균 환경에서와 세중에서 다음 단계를 실시한다. 물론 각 멸균 LB + 앰프 / 아라비 노스 국물 (1 MM의 암피실린 / 10 MM의 아라비 노스)의 199 μl를 추가합니다. 세 개의 개별 컨트롤의 접시에 방을 포함합니다. <ol><li> 다음과 같은 세 가지 컨트롤을 사용하여 잘 (1)를 포함하는 LB + 앰프 / 아라비 노스 국물 만 잘 (2) (공백) 변환되지 않은 유능한 세포는 빈 역할을 위해. 정량 (3) 종결 강도 비 종결 서열에 결찰 미가공 원래 PGR 또는 함유 PGR 블루를 함유하는 세포 (정량 기준 제어)을 포함한다. 플레이트 리더 레이아웃 그림 1을 참조하십시오. </li></ol></li><li> 피펫 오에서 세포 용액을 1 μL / 96- 웰 플레이트의 각각의 웰에 N 브로스. </li><li> 통기성 커​​버 인감 접시와 37 ° C에서 18 ~ 20 시간, 160 rpm으로 흔들어. </li><li> 플레이트 리더를 사용하여 읽어 OD (600)를 결정600 nm에서의 흡광도. 다음과 같이 GFP의 형광을 측정 : 여자 (395) 및 배출 (509) 측정 RFP 형광을 다음과 같이 여자 (575) 및 배출 (605)을 18 ~ 20 시간 후, OD (600)는 약 0.5이어야한다. </li><li> 성장에 변화를 정상화하려면 방정식을 사용 : <img alt="식 (1)" src="/files/ftp_upload/54064/54064eq1.jpg" /> GFP 및 RFP 모두. </li><li> 정규화 한 후, 수학 식을 이용하여 상대 터미네이터 강도를 결정 <img alt="식 (2)" src="/files/ftp_upload/54064/54064eq2.jpg" /></li></ol>

<ol>
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The authors have nothing to disclose.

이 프로토콜에서 가장 중요한 단계는 순서에 적절한 올리고 뉴클레오티드 디자인 사전이다. 올리고 뉴클레오티드는 적합한 접착 말단은 GGA의 혼입이 가능하도록 상부 및 하부 두 가닥의 5 &#39;말단에 추가한다. 또한, 오른쪽에 직면의 왼쪽에 직면 터미네이터 (하단 가닥에 전사를 중지 터미네이터)의 방향을 전환하는 것이 중요하다 GFP 및 RFP 발현이 직면 오른쪽에 있기 때문에 (위의 가닥을 터미?...

Large synthetic biology projects necessitate the use of highly effective transcription terminators to help regulate gene expression. Identification of novel terminators requires bioinformatic analysis of novel genomes. However, as increasing amounts of bioinformatic software are developed, each with a unique algorithm utilized for prediction, more discrepancy between putative results occurs. Because this process is somewhat subjective and is done in silico, these predictions need biological confirmation.1 Additionally, the volume of putative terminators identified through in-silico analysis requires the use of cloning strategies that can be completed in a relatively short time frame. 
The PGR-Blue plasmid is a modification of the PGR plasmid that has been redesigned to use Golden Gate Assembly (GGA) to simplify the cloning procedure by allowing for all reaction steps to be simultaneously performed in one micro-centrifuge tube.2,3 Color selection was incorporated into the plasmid to increase the ease of identifying positive colonies. A successful ligation should be white/yellow in visible light and fluoresce green under blue (450 nm) or ultraviolet (UV) light when grown on plates containing arabinose. Because uncut pGR-blue contains a blue chromo protein (amilCP), colonies containing an unmodified plasmid are blue under visible light. This simplification along with the streamlined protocol allows researchers to proceed from bioinformatic identification to biological confirmation in three to four days. The design nature of this system can be beneficial both in the research lab and in educational settings.
The pGR-Blue plasmid allows for quantification of terminator strength.4 A single arabinose inducible promoter is used to produce green fluorescent protein (GFP) and red fluorescent protein (RFP). The terminator is cloned into the plasmid after the GFP sequence but before the RFP sequence, thus stopping the transcription of the RFP protein. The strength of the terminator is determined by the ratio of GFP produced to RFP produced. 
The Vision and Change5 report suggested that Science, Technology, Engineering and Math (STEM) education incorporate research based experiences into the classroom.6 However, this requires the development of protocols that can be done by students with limited skill sets in a defined time frame. While the protocol can be accomplished in as little as three days, it was also designed so that each major step could be accomplished in a separate weekly (2-3 hr) lab period to create a Course Research Experience (CRE). When used in this manner, the procedure will take between three and six weeks and is appropriate for both introductory and advanced courses in Genetics, Cell Biology or Bioinformatics.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pGR-Blue Plasmid</td>
			<td style="width:243px;">Addgene</td>
			<td style="width:115px;">68374</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGR-Plasmid</td>
			<td>Addgene</td>
			<td style="width:115px;">46002</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AeraSeal-(Sterile Sheets)</td>
			<td>Excel Scientific</td>
			<td>BS-25</td>
			<td style="width:235px;">Sterile Sheets only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X T4 DNA ligase Buffer</td>
			<td>NEB</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BsaI-HF</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td style="width:235px;">The non-HF enzyme will work but is less heat stable.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">NEB Golden Gate Assembly Mix</td>
			<td>NEB</td>
			<td>E1600S</td>
			<td style="width:235px;">Commerial Master Mix refered to in the protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Microcentrifuge Floating Rack</td>
			<td>Nova Tech International</td>
			<td>F18875-6401</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A-3256</td>
			<td style="width:235px;">D-Arabinose will not induce the pBAD promoter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Broth, Miller</td>
			<td>Sigma Aldrich</td>
			<td>L1900</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Agar , Miller&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L2025</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tecan-Infinite M200 Plate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mix &#38; Go Competent Cells - Strain JM109</td>
			<td>Zymo Research</td>
			<td>T3005</td>
			<td style="width:235px;">Use company recommended transformation protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ApE: A plasmid editor-software</td>
			<td style="width:243px;">http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl, Molecular Grade</td>
			<td>Promega</td>
			<td>H5121</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Sodium Chloride (Crystalline/Biological, Certified)</td>
			<td>Fisher Chemical</td>
			<td>S671</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Comercial Oligonucleotide synthesis</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td style="width:235px;">http://www.idtdna.com/site</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Microtest Tissue Culture Plates- 96 well (Sterile)</td>
			<td>Falcon</td>
			<td>35-3072</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mycobacteriophage &#34;Bernal13&#34;</td>
			<td>Genebank</td>
			<td>KJ510413</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>IDT004</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, L-shaped Hockey-Stick Cell</td>
			<td>Life Science Products</td>
			<td>6444-S1</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nano-Drop 2000c UV-Vis Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>2000c</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td>
			<td style="width:243px;">http://rna.igmors.u-psud.fr/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
	</tbody>
</table>

rapid verification of terminators using the pgr-blue plasmid and golden gate assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

이 프로토콜은 골든 게이트 조립 (그림 2)를 사용하여 GFP 및 RFP 사이에 결찰 터미네이터와 PGR - 블루를 포함하는 세포를 생성합니다. 결찰 삽입물을 함유하는 포지티브 콜로니 색에 기초하여 선택 될 수있다. 가시 광선에서 긍정적 인 식민지 / 흰색, 노란색되며 오탐 (false positive)은 37 ° C (그림 3)에서 배양의 18 ~ 20 시간 후 푸른 식민지를 생성합니?...

Watch this Scientific Journal Video about Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly at JoVE.com

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

PGR - 블루 플라스미드와 골든 게이트 어셈블리를 사용하여 터미네이터의 신속한 확인

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed ...

rapid-verification-terminators-using-pgr-blue-plasmid-golden-gate

Research

JoVE Journal

Biology

7.6K 조회수.  Ouachita Baptist University. This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

비디오: Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

접합에 참여 전송 단백질의 서열 별 분석을위한 공역 결합 분석 실험

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

연구

유전학

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

대규모 gRNA DNA 소스에서 라이브러리 제작에 대 한 범용 프로토콜

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

단백질 상호 작용을 비교 하는 배치에 효 모 2 잡종 스크린

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

생화학

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

유도할 수 있는, 애기 thaliana 통해 LhGR 중재 트랜스에 셀 형식 관련 식-활성화

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

모듈형 골든 게이트 복제를 사용하여 다중 유전자 구조물의 신속한 조립

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

생체공학

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

디자이너 클레아 제를 사용하여 마우스 게놈 엔지니어링

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

생물학

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

플라스미드 벡터를 생성 깁슨 어셈블리를 사용하여 인간의 신장 요인-1α의 발기인의 규정에 따라 FLAG-태그 단백질을 표현

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

플라스미드 유래 구현 화학 반응 네트워크 용 DNA 가닥 변위 게이츠

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

높은 처리량 시퀀싱을 활용하여 전체 - 단백질 포화 돌연변이 유발 도서관의 기능 평가를위한 프로토콜

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...