丝状蓝藻腔隙的自然转化、蛋白表达和低温保存
Summary
Phormidium lacuna 是一种从海洋岩石池中分离出来的丝状蓝藻。本文介绍了从天然来源中分离细丝，DNA提取，基因组测序，自然转化，sfGFP表达，冷冻保存和运动性方法。
Abstract
蓝藻是基础研究和生物技术项目的重点，其中太阳能用于生物质生产。 Phormidium lacuna 是一种新分离的丝状蓝藻。本文描述了如何从海洋岩石池中分离出新的丝状蓝藻。它还描述了如何从细丝中提取DNA以及如何对基因组进行测序。虽然许多单细胞物种的转化已经建立，但丝状蓝藻的报道较少。这里描述了一种简化的 P. lacuna 自然转化方法。 P. lacuna 是Oscillatoriales目中唯一建立自然转化的成员。本文还展示了如何使用自然转化来表达超折叠绿色荧光蛋白（sfGFP）。内源性 cpcB 启动子诱导的表达强度约为 cpc560、 A2813 或 psbA2 启动 子的 5 倍。此外，建立了冷冻保存 P. lacuna 和 Synechocystis sp.CPP 6803的方法，并描述了评估液体介质中以及琼脂和塑料表面上的运动性的方法。
Introduction
蓝藻是利用光合作用作为能量来源的原核生物1，2。研究越来越关注蓝藻物种。几种蓝藻可以用DNA3转化。基因可以在这些物种中被敲除或过度表达。然而，转化仅限于少数物种4，5，6，7，8，9，10，11，并且很难在培养品或野生8的品系中建立转化。从海洋岩石池中分离出丝状物种 Phormidium lacuna 的菌株（图1），其中环境条件（例如盐浓度或温度）随时间波动。这些丝状蓝藻可以用作它们所属的Oscillatoriales12 目模式生物。
在试验测试通过电穿孔13，14进行基因转移时，发现 P. lacuna 可以通过自然转化15转化。在这个过程中，DNA被一些细胞自然吸收。与其他转化方法16，17相比，自然转化的优点是不需要可能使过程复杂化的附加工具。例如，电穿孔需要适当的比色皿，完整的电线和选择适当的电压。 P. lacuna 是目前唯一易受自然转化影响的振荡器成员。由于原始方案基于电穿孔方案，因此它仍然包括几个可能不必要的洗涤步骤。测试了不同的方法来简化协议，从而产生了这里介绍的转换协议。
基因组序列对于基于基因敲除或过表达的进一步分子研究至关重要。虽然基因组序列可以在短时间内用下一代测序机获得，但DNA的提取可能很困难，并且取决于物种。对于 P. lacuna，测试了几种方案。然后建立了基于改良的十六烷基三甲基溴化铵（CTAB）的方法，从而产生了每个纯化循环的DNA纯度和DNA产量的可接受纯度，以便在实验室中继续工作。可以使用该协议对五种菌株的基因组进行测序。下一个合乎逻辑的转化步骤是在 P. lacuna中建立蛋白质表达。
在该协议中用作标记蛋白的sfGFP可以用任何荧光显微镜检测。所有测试的启动子都可用于 P. lacuna sfGFP表达。转化产生的菌株数量不断增加，导致需要一种储存培养物的方法。这些方法已针对 大肠杆菌 和许多其他细菌18建立。在标准方案中，制备甘油培养物，在液氮中转移，并在-80°C下储存。 这种方法只需要几个步骤，对于那些建立它的物种来说非常可靠。标准方案对于 P. lacuna 是不可行的，因为活细胞不能在所有情况下都恢复。然而，当甘油在解冻后被除去时，所有试验的细胞都存活了下来。提出了用于分析 P. lacuna运动性的简单方法，其可以与敲除诱变相结合以研究IV型舂毛或光感受器的作用。这些测定与单细胞蓝藻19，20，21 的测定不同，并且也可用于其他Oscillatoria。
Protocol
1. 与自然环境隔绝 注意：可以分离绿藻，硅藻，丝状蓝藻和其他微藻。该方案可用于在实验室条件下生长的岩池中的任何微藻物种。属于Oscillatoriales的丝状蓝藻可以通过其运动和丝状形状轻松识别。该物种可以通过基因组测序或16S rRNA测序在半纯状态下鉴定。 将来自海洋岩石池（即岩石海岸的空腔）的液态海水样品转移到50 mL烧瓶中。对于每个烧瓶，请注…
Representative Results
按照上述方法，从岩池中分离出5种不同的 P. lacuna 菌株并测序（图1 和 表1）。除 P. lacuna HE10JO外，所有培养物在传代培养约1年后均无菌。该菌株仍然受到海洋细菌 Marivirga atlantica的污染。在随后的黑尔戈兰岛游览中，从岩石池中分离出其他丝状蓝藻，这些蓝藻与 P. lacuna 不同，需要表征。 对几种DNA提取和纯?…
Discussion
尽管许多蓝藻菌株可从培养物32，33，34，35，36中获得，但由于这些物种适应特定特性，因此仍需要来自野生的新蓝藻。从岩池中收集 P. lacuna ，并适应盐浓度和温度30的变化。该物种的菌株在2008年，2009年和2010年的短途旅行中被发现。通过这里描述的…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了卡尔斯鲁厄理工学院的支持。
Materials
| Autoclave 3870 ELV | Tuttnauer | 3870 ELV | |
| Bacto Agar | OttoNorwald | 214010 | |
| BG-11 Freshwater Solution | Sigma Aldrich | C3061 | |
| BG-11 medium | Merck | 73816-250ML | |
| Boric acid | Merck | 10043-35-3 | H3BO3 |
| Calcium chloride dihydrate | Carl Roth | 10035-04-8 | CaCl2 · 2 H2O |
| Cell culture flasks Cellstar with filter screw cap, sterile, 250 mL | Greiner | 658190 | |
| Cell culture flasks Cellstar with filter screw cap, sterile, 50 mL | Greiner | 601975 | |
| Centrifuge LYNX 4000 | Thermo Scientific | 75006580 | and rotor |
| Centrifuge microstar 17 | VWR International | N/A | for up to 13,000 rpm |
| Cetyltrimethylammonium Bromide (CTAB) | PanReac AppliChem | 57-09-0 | C19H42BrN |
| Chloroform : Isoamyl Alcohol 24 : 1 | PanReac AppliChem | A1935 |
|
| Cobalt(II) chloride hexahydrate | Merck | 7791-13-1 | CoCl2 · 6 H2O |
| Copper(II) sulphate pentahydrate | Merck | 7758-99-8 | CuSO4 · 5 H2O |
| D(+)-Biotin | Carl Roth | 58-85-5 | C10H16N2O3S |
| DNA ladder 1 kb | New England Biolabs | N3232 | |
| DNA ladder 100 bp | New England Biolabs | N3231 | |
| Electrical pipetting help accujet-pro S | Brand GmbH | 26360 | for pipetting 1-25 mL |
| Ethanol | VWR | 64-17-5 | C2H6O |
| Ethylenediamine tetraacetic acid disodium salt dihydrate | Carl Roth | 6381-92-6 | EDTA-Na2 · 2 H2O |
| Fluorescence microscope ApoTome | Zeiss | ||
| Fluorescence microscope Axio Imager 2 | Zeiss | ||
| French Pressure Cell Press | American Instrument Company | N/A | |
| Gel documation System Saffe Image | Invitrogen | ||
| Gelelctrophoresis system Mupid-One/-exu | ADVANCED | ||
| Glassware, different | |||
| Glycerol | Carl Roth | 56-81-5 | C3H8O3 |
| Iron(III) chloride hexahydrate | Merck | 10025-77-1 | FeCl3 · 6 H2O |
| Kanamycin | Sigma-Aldrich | 25389-94-0 | |
| Kanamycin sulphate | Carl Roth | 25389-94-0 | C18H36N4O11 · H2SO4 |
| Lauroylsarcosine, Sodium Salt (Sarcosyl) | Sigma Aldrich | 137-16-6 | C15H28NO3 · Na |
| LB Broth (Lennox) | Carl Roth | X964.4 | |
| Light source, fluorescent tube L18W/954 daylight | OSRAM | cultivation of cyanobacteria | |
| Light source, LED panel XL 6500K 140 W | Bloom Star | N/A | cultivation of cyanobacteria, up to 1,000 µmol m-2 s-1 |
| Magnesium chloride hexahydrate | Carl Roth | 7791-18-6 | MgCl2 · 6 H2O |
| Manganese(II) chloride tetrahydrate | Serva | 13446-34-9 | MnCl2 · 4 H2O |
| Microscope DM750 | Zeiss | ||
| Midi prep plasmid extraction kit NucleoBond Xtra Midi kit | Macherey-NAGEL GmbH & Co. KG | REF740410.50 | |
| Minicomputer Raspberry Pi 4 + | Conrad Electronics | 2138863-YD | for time-lapse recording |
| Ocular camera EC3 | Leica | for continuous recording up to 30 s | |
| Ocular camera MikrOkular Full HD | Bresser | for time-lapse recordings, coupled to Raspberry Pi minicomputer | |
| Petri dishes polystyrole, 100 mm x 20 mm | Merck | P5606-400EA | |
| Petri dishes polystyrole, 60 mm x 15 mm | Merck | P5481-500EA | |
| Photometer Nanodrop ND-1000 | Peqlab Biotechnologie | ||
| Photometer Uvikon XS | Goebel Instrumentelle Analytik GmbH | ||
| Pipetman 100-1,000 µL | Gilson | SKU: FA10006M | |
| Pipetman 10-100 µL | Gilson | SKU: FA10004M | |
| Plastic pipettes 10 mL, sterile | Greiner | 607107 | |
| Plastic tube, sterile, 15 mL | Greiner | 188271 | |
| Plastic tube, sterile, 50 mL | Greiner | 227261 | |
| Potassium bromide | Carl Roth | 7758-02-3 | KBr |
| Potassium chloride | Carl Roth | 7447-40-7 | KCl |
| Power supply Statron 3252-1 | Statron Gerätetechnik GmbH | ||
| Power supply Voltcraft PPS 16005 | Conrad Electronics | for LED | |
| Proteinase K | Promega | MC500C | from Maxwell 16 miRNA Tissue Kit AS1470 |
| Q5 polymerase | New England Biolabs | M0491S | |
| Sequencing kit NextSeq 500/550 v2.5 | Illumina | ||
| Sequencing system NextSeq 550 SY-415-1002 | Illumina | ||
| Shaker Unimax 2010 | Heidolph Instruments | for cultivation | |
| Sodium acetate | Carl Roth | 127-09-3 | NaCH3COO |
| Sodium chloride | Carl Roth | 7647-14-5 | NaCl |
| Sodium dihydrogen phosphate monohydrate | Carl Roth | 10049-21-5 | NaH2PO4 · H2O |
| Sodium fluoride | Carl Roth | 7681-49-4 | NaF |
| Sodium hydrogen carbonate | Carl Roth | 144-55-8 | NaHCO3 |
| Sodium molybdate dihydrate | Serva | 10102-40-6 | Na2MoO4 · 2 H2O |
| Sodium nitrate | Merck | 7631-99-4 | NaNO3 |
| Sodium sulphate | Carl Roth | 7757-82-6 | Na2SO4 |
| Strontium chloride hexahydrate | Carl Roth | 10025-70-4 | SrCl2 · 6 H2O |
| Thiamine hydrochloride | Merck | 67-03-8 | C12H17ClN4OS · HCl |
| TRIS | Carl Roth | 77-86-1 | C4H11NO3 |
| Ultrasonic device UP100H with sonotrode MS3 | Hielscher Ultrasound Technology | UP100H | |
| Ultraturrax Silent Crusher M | Heidolph Instruments | homogenizer | |
| Urea | Carl Roth | 57-13-6 | CH4N2O |
| Vitamin B12 | Sigma | 68-19-9 | C63H88CoN14O14P |
| Vitamin solution | 0.3 µM thiamin-HCl, 2.1 nM biotin, 0.37 nM cyanocobalamin | ||
| Water Stills, Water treatment | VEOLIA water technologies | ELGA_21001 | |
| Zinc sulphate heptahydrate | Sigma | 7446-20-0 | ZnSO4 · 7 H2O |
| software, URL | |||
| gatb-minia program for DNA assembly | https://github.com/GATB/gatb-minia-pipeline | makes large scaffolds from short DNA reads, Linux based | |
| ImageJ | software for immage processing (pixel intensities, circle diameter) | ||
| RAST annotation server | https://rast.nmpdr.org | input: genome DNA sequence, detects open reading frames, lists protein sequences and their functions | |
| Culture media | |||
| Artificial seawater | 0.41 M NaCl , 53 mM MgCl2,28 mM Na2SO4, 10 mM CaCl2 , 9 mM KCl , 2.4 mM NaHCO3 ,0.84 mM KBr, 0.49 mM H3BO3, 90 µM SrCl2, 72 µM NaF | ||
| f/2 -liquid medium | artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution, 0.88 mM NaNO3, 36 µM NaH2PO4 | ||
| f/2+ liquid medium | f/2-medium, with 10 times increased NaNO3 and NaH2PO4 (0.88 mM NaNO3, 36 µM NaH2PO4 | ||
| f/2+-agar | 3 % (w/v) bacto agar, artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution ,8.8 mM NaNO3, 0.36 mM NaH2PO4 | ||
| f/2-agar | 3 % (w/v) bacto agar, artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution ,0.88 mM NaNO3, 36 µM NaH2PO4 | ||
| Trace element solution | 0.36 mM NaH2PO4, 12 µM Na2EDTA, 39 nM CuSO4, 26 nM Na2MoO4 , 77 nM ZnSO4, 42 nM CoCl2, 0.91 µM MnCl2 | ||
| Vitamin solution | 0.3 µM thiamin-HCl, 2.1 nM biotin, 0.37 nM cyanocobalamin | ||
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Diesen Artikel zitieren
Weber, N., Hofmeister, M., Wunsch, N., Kohler, A., Kaster, A., Vollmers, J., Kachel, B., Mack, M., Lamparter, T. Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna. J. Vis. Exp. (180), e63470, doi:10.3791/63470 (2022).