网站特定的细菌染色体工程：ΦC31整合介导的盒式交换（IMCE）
Summary
描述一个快速和有效的方法融入预制受体菌株，称为停机坪株，外源DNA的利益。该方法允许进入工程着陆垫一个给定的应变轨迹的站点特定的DNA盒的整合，通过共轭ΦC31整合表达。
Abstract
可用于细菌染色体的稳定保持在大型基地范围内1外源DNA。整合到染色体复制质粒，质粒稳定性，质粒不相容性和质粒拷贝数变异，如规避的问题。这种方法使用从链霉菌噬菌体（Φ）C31的2,3特定地点的整合。 ΦC31整合酶催化两个特定的DNA位点之间的直接重组：attB和attP（34和39个基点，分别）4。此重组是稳定的，并没有恢复5。一个“着陆垫”（LP）的1霉素抗性基因，AADA（SPR），和大肠杆菌组成的序列大肠杆菌 β-葡萄糖醛酸酶基因（uidA基因 ）两侧attP网站已整合到苜蓿根瘤菌，Ochrobactrum anthropi，在区域间的农杆菌染色体， 上午PC轨迹和的TETA轨迹，分别与根瘤菌用于在该协议。捐助动员的载体侧翼1填塞红色荧光蛋白（RFP）基因和抗生素抗性基因的attB网站也已建成。在这个例子中使用庆大霉素的耐药质粒pJH110。使用SPH我和 Pst一所需的结构可能会被替换的RFP基因6另外attB网站两侧，可能是一种人工合成的构造子克隆到动员的载体，如pK19mob 7。由lac启动子驱动的ΦC31整合（克隆pHS62 8）基因的表达质粒pRK7813 9上动员的广泛的寄主范围。
一个tetraparental交配协议用于转移到捐助盒的LP菌株，从而取代捐助卡带在LP序列标记。这些细胞的移植S-稳定整合。横贯稳定整合，形成了0.5％的典型效率。横贯稳定整合通常是发现在第一500-1000菌落筛选抗生素的敏感性，或用5 – 溴-4 – 氯-3 – 吲哚基-β-D-葡萄糖醛酸（X GLUC）蓝白筛选。该协议包含用于创建和隔离跨稳定整合的交配和选拔程序。
Protocol
1。文化生产准备无菌液体介质：TY 10（5克/ L，蛋白胨，酵母提取物3克/升，0.44克/升的氯化钙脱水），11磅（10克/ L，蛋白胨，5 g / L酵母提取物，5克/氯化钠，pH值7）。 从一个单一的殖民地：TY传媒SmUW227（ 根瘤菌的 LP应变：建设将在别处描述，紧张施工细节要求）（）到5毫升50微克/毫升大观接种。接种以下株，到5毫升的LB液体培养基，给予抗生素补充： ?…
Discussion
该的IMCE技术允许为一个单一的attB两侧LP轨迹以前工程菌的DNA磁带的有效整合。一旦RFP创建捐助盒克隆所需的构造，该技术并不需要随后的DNA纯化和改造，使得它非常健壮。这是至关重要的，包括适当的生长控制，是某些抗生素耐药性是由于建立反稳定整合，而不是其他因素。
IMCE生产效率约0.5％的反式稳定整合。相反，通过同源重组的双重交叉发生在约10 -6?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
请提供整合克隆玛格丽特厘米史密斯
资金支持：
加拿大基因组/基因组大草原
NSERC发现和战略项目赠款
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Streptomycin | Bioshop Canada Inc. | STP101 | |
| Spectinomycin | Bioshop Canada Inc. | SPE201 | |
| Gentamicin | Bioshop Canada Inc. | GTA202 | |
| Choramphenicol | Bioshop Canada Inc. | CLR201 | |
| Tetracycline | Bioshop Canada Inc. | TET701 | |
| Kanamycin | Bioshop Canada Inc. | KAN201 | |
| Bacteriological grade agar | Bioshop Canada Inc. | AGR001 | |
| Tryptone | Bioshop Canada Inc. | TRP402 | |
| Yeast Extract | Bioshop Canada Inc. | YEX401 | |
| Sodium Chloride | Bioshop Canada Inc. | SOD001 | |
| Calcium Chloride | Bioshop Canada Inc. | CCL444 | |
| X-gluc | Gold Biotechnology Inc. | G1281C1 | |
| E. coli MT616 strain | Available upon request | Also used outside of our lab | |
| E. coli pJC2 strain | In house, available by request | ||
| E. coli pJH110 strain | In house, available by request | ||
| SmUW227 strain | In house, available by request |
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Tags
Site-specificBacterial Chromosome EngineeringΦC31 IntegraseCassette ExchangeAttBAttPRecombinationLanding PadSpectinomycin-resistance GeneAadAE. Coli ß-glucuronidase GeneUidASinorhizobium MelilotiOchrobactrum AnthropiAgrobacterium TumefaciensAmpC LocusTetA LocusMobilizable Donor VectorsStuffer Red Fluorescent Protein (rfp) GeneAntibiotic Resistance GeneGentamicin Resistant Plasmid PJH110SphIPstI
Cite This Article
Heil, J. R., Cheng, J., Charles, T. C. Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE). J. Vis. Exp. (61), e3698, doi:10.3791/3698 (2012).