在型号蓝藻种类标记和无标记突变体的产生
Summary
Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.
Abstract
Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of ‘escape’ of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.
Introduction
蓝藻是地球上几乎每一个自然环境中的细菌的进化古老而多样化的门。在海洋生态系统中，他们都特别丰富，许多营养循环中发挥关键作用，约占一半的固碳1，在海洋中的大多数固氮2和数百万吨的油气产量3每年。叶绿体，细胞器负责在真核藻类和植物的光合作用，很可能已经从由宿主生物体4吞噬蓝藻演变。蓝藻已经证明是有用的模式生物进行光合作用，电子传递5和生化途径，其中许多植物中是保守的研究。此外蓝藻越来越多地被用于生产食品，生物燃料6，电力7和工业化合物8，由于它们的喜的水和CO 2 ghly有效转化生物质使用太阳能9。许多物种可以在非耕地用最少的营养物和海水中培养，这表明蓝藻可能在大规模生长，而不会影响农业生产。某些物种也是天然产品，包括抗真菌剂，抗菌剂和抗癌化合物10,11的源。
产生突变的能力是关键了解蓝藻光合作用，生物化学和生理学，以及必不可少的菌株用于工业用途的发展。大多数已发表的研究中产生遗传上通过抗生素抗性盒的插入修饰的菌株到感兴趣的部位。这限制了可被引入到菌株，因为只有少数的抗生素抗性盒可用于在蓝藻使用突变的数量。含有基因的菌株抗生素赋予重sistance不能用于工业生产中的开放的池塘，这很可能是唯一的成本效益的方法，以产生生物燃料和其它低价值产品12。未标记突变体的产生克服了这些限制。未标记的突变体不包含外源DNA的，除非有意包括在内，并且可以操作多次。因此，可以根据需要在一个菌株，以产生尽可能多的改变。此外，在变形例的网站的下游基因极性影响可以最小化，从而使机体13的更精确的修饰。
为了产生突变体菌株，自杀质粒在蓝藻染色体含有两个DNA片段相同的区域侧翼的基因被删除（称为5'和3'侧翼区）的第一构造。然后两个基因这些侧翼区域之间插入。其中的一个编码抗生素抗性蛋白;第二编码将SacB，这督促集体企业果聚糖蔗糖酶的化合物，赋予蔗糖敏感性。在此过程中的第一阶段中，标记的突变体，含有一些外源DNA 即菌株产生。该质粒构建体与蓝藻细胞混合和该DNA是由生物体吸收自然。转化体是由生长在含有适当的抗生素和通过PCR验证了突变体基因型琼脂平板上选择。自杀质粒不能感兴趣的菌株中复制。因此，任何抗生素抗性菌落，将导致从由此感兴趣的基因中插入染色体中的重组事件。以产生未标记突变体中，明显的突变，然后用仅包含5'和3'侧翼区的第二自杀质粒混合。然而，如果需要的外源DNA的插入，由5'和3'侧翼用含有这些DNA片段之间插入目的基因的盒的区域的质粒，可以使用。塞莱ction是，通过含有蔗糖琼脂平板上生长。作为当sacB基因产物被表达蔗糖是致死的细胞，该存在的唯一的细胞是其中已经发生了第二次重组事件，由此，蔗糖敏感性基因，除了在抗生素抗性基因，已被重组出的染色体上的质粒。作为重组交换的结果，侧翼区和它们之间的任何DNA被插入到染色体。
我们已经成功地使用这些方法来产生在集胞藻的同一菌株的多个染色体突变。 6803（以下简称为集胞藻属 ）13,14，引入单点突变到感兴趣13的基因和用于基因盒的表达。而代无标记击倒之前已经提供了工作在集胞藻 15,16，详细的方法证实，由辅助的关键步骤的视觉呈现，是不公开的。我们还以另一种模式蓝藻， 聚球藻申请代明显击倒同样的方法。 PCC7002（以下简称为聚球藻 ）。该协议提供了产生突变体和用于验证和存储这些菌株快速协议的清晰，简单的方法。
Protocol
Representative Results
Discussion
在无人盯防的产生突变体的最重要的步骤是：1）精心设计载体，以确保只有目标区域被改变; 2）确保样品保持无菌，尤其是当上培养的蔗糖; 3）电镀转化最初在缺乏抗生素，随后加入琼脂加抗生素24小时后的BG11琼脂平板上标注的突变体产生的细胞; 4）培养标志着前4整天突变体上镀BG11加蔗糖琼脂平板：5）确保标记突变体是完全隔离和6）确认彻底突变株的基因型。对于这最后的步骤中，设计用于?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢环境服务协会教育信托基金，在剑桥SynBio基金合成生物学和社会正义和赋权，印度政府部，给予资金支持。
Materials
| NaNO3 | Sigma | S5506 | |
| MgSO4.7H2O | Sigma | 230391 | |
| CaCl2 | Sigma | C1016 | |
| citric acid | Sigma | C0759 | |
| Na2EDTA | Fisher | EDT002 | |
| H3BO3 | Sigma | 339067 | |
| MnCl2.4H2O | Sigma | M3634 | |
| ZnSO4.7H2O | Sigma | Z4750 | |
| Na2MoO4.2H2O | Sigma | 331058 | |
| CuSO4.5H2O | Sigma | 209198 | |
| Co(NO3)2.6H2O | Sigma | 239267 | |
| Ferric ammonium citrate | Sigma | F5879 | |
| K2HPO4 | Sigma | P3786 | |
| Na2CO3 | Fisher | SODC001 | |
| TES | Sigma | T1375 | |
| NaHCO3 | Fisher | SODH001 | |
| HEPES | Sigma | H3375 | |
| cyanocobalamin | Sigma | 47869 | |
| Na2S2O3 | Sigma | 72049 | |
| Bacto agar | BD | 214010 | |
| Sucrose | Fisher | SUC001 | |
| Petri dish 90 mm triple vented | Greiner | 633185 | |
| 0.2 µm filters | Sartorius | 16534 | |
| 100 mL conical flasks | Pyrex | CON004 | |
| Parafilm M 100 mm x38 m | Bemis | FIL003 | |
| Phusion high fidelity DNA polymerase | Phusion | F-530 | |
| Agarose | Melford | MB1200 | |
| DNA purification kit | MoBio | 12100-300 | |
| Restriction endonucleases | NEB | ||
| T4 ligase | Thermo Scientific | EL0011 | |
| Luria Bertani broth | Invitrogen | 12795-027 | |
| MES | Sigma | M8250 | |
| Kanamycin sulfate | Sigma | 60615 | |
| Ampicillin | Sigma | A9518 | |
| GeneJET plasmid miniprep kit | Thermo Scientific | K0503 | |
| 14 mL round-bottom tube | BD falcon | 352059 | |
| GoTaq G2 Flexi DNA polymerase | Promega | M7805 | |
| 425-600 µm glass beads | Sigma | G8772 | |
| Glycerol | Sigma | G5516 | |
| DMSO | Sigma | D8418 | |
| Fluorescent bulbs | Gro-Lux | 69 | |
| HT multitron photobioreactor | Infors |
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