Name: natural transformation, protein expression, and cryoconservation of the filamentous cyanobacterium phormidium lacuna
Uploaded: 2022-02-01T14:30:00
Description: Watch this Scientific Journal Video about Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna at JoVE.com

这项工作得到了卡尔斯鲁厄理工学院的支持。

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。
<ol><li>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...

<ol>
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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclave 3870 ELV</td>
			<td>Tuttnauer</td>
			<td>3870 ELV</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>OttoNorwald</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>BG-11 Freshwater Solution</td>
			<td>Sigma Aldrich</td>
			<td>C3061</td>
			<td></td>
		</tr>
		<tr>
			<td>BG-11 medium</td>
			<td>Merck</td>
			<td>73816-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Merck</td>
			<td>10043-35-3</td>
			<td>H3BO3</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Carl Roth</td>
			<td>10035-04-8</td>
			<td>CaCl2 &#183; 2 H2O</td>
		</tr>
		<tr>
			<td>Cell culture flasks Cellstar with filter screw cap, sterile, 250 mL</td>
			<td>Greiner</td>
			<td>658190</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks Cellstar with filter screw cap, sterile, 50 mL</td>
			<td>Greiner</td>
			<td>601975</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge LYNX 4000</td>
			<td>Thermo Scientific</td>
			<td>75006580</td>
			<td>and rotor</td>
		</tr>
		<tr>
			<td>Centrifuge microstar 17</td>
			<td>VWR International</td>
			<td>N/A</td>
			<td>for up to 13,000 rpm</td>
		</tr>
		<tr>
			<td>Cetyltrimethylammonium Bromide (CTAB)</td>
			<td>PanReac AppliChem</td>
			<td>57-09-0</td>
			<td>C19H42BrN</td>
		</tr>
		<tr>
			<td>Chloroform : Isoamyl Alcohol 24 : 1</td>
			<td>PanReac AppliChem</td>
			<td> 
			A1935</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt(II) chloride hexahydrate</td>
			<td>Merck</td>
			<td>7791-13-1</td>
			<td>CoCl2 &#183; 6 H2O</td>
		</tr>
		<tr>
			<td>Copper(II) sulphate pentahydrate</td>
			<td>Merck</td>
			<td>7758-99-8</td>
			<td>&#160;CuSO4 &#183; 5 H2O</td>
		</tr>
		<tr>
			<td>D(+)-Biotin</td>
			<td>Carl Roth</td>
			<td>58-85-5</td>
			<td>&#160;C10H16N2O3S</td>
		</tr>
		<tr>
			<td>DNA ladder 1 kb</td>
			<td>New England Biolabs</td>
			<td>N3232</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA ladder 100 bp</td>
			<td>New England Biolabs</td>
			<td>N3231</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical pipetting help accujet-pro S</td>
			<td>Brand GmbH</td>
			<td>26360</td>
			<td>for pipetting 1-25 mL</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>64-17-5</td>
			<td>C2H6O</td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid disodium salt dihydrate</td>
			<td>Carl Roth</td>
			<td>6381-92-6</td>
			<td>EDTA-Na2 &#183; 2 H2O</td>
		</tr>
		<tr>
			<td>Fluorescence microscope ApoTome</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope Axio Imager 2</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>French Pressure Cell Press</td>
			<td>American Instrument Company</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel documation System Saffe Image</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelelctrophoresis system Mupid-One/-exu</td>
			<td>ADVANCED</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glassware, different</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth</td>
			<td>56-81-5</td>
			<td>C3H8O3</td>
		</tr>
		<tr>
			<td>Iron(III) chloride hexahydrate</td>
			<td>Merck</td>
			<td>10025-77-1</td>
			<td>&#160;FeCl3 &#183; 6 H2O</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>25389-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin sulphate</td>
			<td>Carl Roth</td>
			<td>25389-94-0</td>
			<td>C18H36N4O11 &#183; H2SO4</td>
		</tr>
		<tr>
			<td>Lauroylsarcosine, Sodium Salt (Sarcosyl)</td>
			<td>Sigma Aldrich</td>
			<td>137-16-6</td>
			<td>C15H28NO3 &#183; Na</td>
		</tr>
		<tr>
			<td>LB Broth (Lennox)</td>
			<td>Carl Roth</td>
			<td>X964.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Light source, fluorescent tube L18W/954 daylight</td>
			<td>OSRAM</td>
			<td></td>
			<td>cultivation of cyanobacteria</td>
		</tr>
		<tr>
			<td>Light source, LED panel XL 6500K 140 W</td>
			<td>Bloom Star</td>
			<td>N/A</td>
			<td>cultivation of cyanobacteria, up to 1,000 &#181;mol m-2 s-1</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Carl Roth</td>
			<td>7791-18-6</td>
			<td>MgCl2 &#183; 6 H2O</td>
		</tr>
		<tr>
			<td>Manganese(II) chloride tetrahydrate</td>
			<td>Serva</td>
			<td>13446-34-9</td>
			<td>MnCl2 &#183; 4 H2O</td>
		</tr>
		<tr>
			<td>Microscope DM750</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Midi prep plasmid extraction kit NucleoBond Xtra Midi kit</td>
			<td>Macherey-NAGEL GmbH &#38; Co. KG</td>
			<td>REF740410.50</td>
			<td></td>
		</tr>
		<tr>
			<td>Minicomputer Raspberry Pi 4 +</td>
			<td>Conrad Electronics</td>
			<td>2138863-YD</td>
			<td>for time-lapse recording</td>
		</tr>
		<tr>
			<td>Ocular camera EC3</td>
			<td>Leica</td>
			<td></td>
			<td>for continuous recording up to 30 s</td>
		</tr>
		<tr>
			<td>Ocular camera MikrOkular Full HD</td>
			<td>Bresser</td>
			<td></td>
			<td>for time-lapse recordings, coupled to Raspberry Pi minicomputer</td>
		</tr>
		<tr>
			<td>Petri dishes polystyrole, 100 mm x 20 mm</td>
			<td>Merck</td>
			<td>P5606-400EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes polystyrole, 60 mm x 15 mm</td>
			<td>Merck</td>
			<td>P5481-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer Nanodrop ND-1000</td>
			<td>Peqlab Biotechnologie</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer Uvikon XS</td>
			<td>Goebel Instrumentelle Analytik GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman 100-1,000 &#181;L</td>
			<td>Gilson</td>
			<td>SKU: FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman 10-100 &#181;L</td>
			<td>Gilson</td>
			<td>SKU: FA10004M</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic pipettes 10 mL, sterile</td>
			<td>Greiner</td>
			<td>607107</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tube, sterile, 15 mL</td>
			<td>Greiner</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tube, sterile, 50 mL</td>
			<td>Greiner</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium bromide</td>
			<td>Carl Roth</td>
			<td>7758-02-3</td>
			<td>KBr</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Carl Roth</td>
			<td>7447-40-7</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Power supply Statron 3252-1</td>
			<td>Statron Ger&#228;tetechnik GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply Voltcraft PPS 16005</td>
			<td>Conrad Electronics</td>
			<td></td>
			<td>for LED</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Promega</td>
			<td>MC500C</td>
			<td>from Maxwell 16 miRNA Tissue Kit AS1470</td>
		</tr>
		<tr>
			<td>Q5 polymerase</td>
			<td>New England Biolabs</td>
			<td>M0491S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing kit NextSeq 500/550 v2.5</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing system NextSeq 550 SY-415-1002</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker Unimax 2010</td>
			<td>Heidolph Instruments</td>
			<td></td>
			<td>for cultivation</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Carl Roth</td>
			<td>127-09-3</td>
			<td>NaCH3COO</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>7647-14-5</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>Carl Roth</td>
			<td>10049-21-5</td>
			<td>NaH2PO4 &#183; H2O</td>
		</tr>
		<tr>
			<td>Sodium fluoride</td>
			<td>Carl Roth</td>
			<td>7681-49-4</td>
			<td>NaF</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Carl Roth</td>
			<td>144-55-8</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium molybdate dihydrate</td>
			<td>Serva</td>
			<td>10102-40-6</td>
			<td>Na2MoO4 &#183; 2 H2O</td>
		</tr>
		<tr>
			<td>Sodium nitrate</td>
			<td>Merck</td>
			<td>7631-99-4</td>
			<td>NaNO3</td>
		</tr>
		<tr>
			<td>Sodium sulphate</td>
			<td>Carl Roth</td>
			<td>7757-82-6</td>
			<td>Na2SO4</td>
		</tr>
		<tr>
			<td>Strontium chloride hexahydrate</td>
			<td>Carl Roth</td>
			<td>10025-70-4</td>
			<td>SrCl2 &#183; 6 H2O</td>
		</tr>
		<tr>
			<td>Thiamine hydrochloride</td>
			<td>Merck</td>
			<td>67-03-8</td>
			<td>C12H17ClN4OS &#183; HCl</td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Carl Roth</td>
			<td>77-86-1</td>
			<td>C4H11NO3</td>
		</tr>
		<tr>
			<td>Ultrasonic device UP100H with sonotrode MS3</td>
			<td>Hielscher Ultrasound Technology</td>
			<td>UP100H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraturrax Silent Crusher M</td>
			<td>Heidolph Instruments</td>
			<td></td>
			<td>homogenizer</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Carl Roth</td>
			<td>57-13-6</td>
			<td>CH4N2O</td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>Sigma</td>
			<td>68-19-9</td>
			<td>C63H88CoN14O14P</td>
		</tr>
		<tr>
			<td>Vitamin solution</td>
			<td></td>
			<td></td>
			<td>0.3 &#181;M thiamin-HCl, 2.1 nM biotin, 0.37 nM cyanocobalamin</td>
		</tr>
		<tr>
			<td>Water Stills, Water treatment</td>
			<td>VEOLIA water technologies</td>
			<td>ELGA_21001</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulphate heptahydrate</td>
			<td>Sigma</td>
			<td>7446-20-0</td>
			<td>ZnSO4 &#183; 7 H2O</td>
		</tr>
		<tr>
			<td>software, URL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gatb-minia program for DNA assembly</td>
			<td>https://github.com/GATB/gatb-minia-pipeline</td>
			<td>makes large scaffolds from short DNA reads, Linux based</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td>software for immage processing (pixel intensities, circle diameter)</td>
			<td></td>
		</tr>
		<tr>
			<td>RAST annotation server</td>
			<td>https://rast.nmpdr.org</td>
			<td>input: genome DNA sequence, detects open reading frames, lists protein sequences and their functions</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial seawater</td>
			<td colspan="3">0.41 M NaCl , 53 mM MgCl2,28 mM Na2SO4, 10 mM CaCl2 , 9 mM&#160; KCl , 2.4 mM NaHCO3 ,0.84 mM KBr, 0.49 mM H3BO3, 90 &#181;M SrCl2, 72 &#181;M NaF</td>
		</tr>
		<tr>
			<td>f/2 -liquid medium</td>
			<td colspan="3">artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution, 0.88 mM NaNO3, 36 &#181;M NaH2PO4&#160;</td>
		</tr>
		<tr>
			<td>f/2+ liquid medium</td>
			<td colspan="3">f/2-medium, with 10 times increased NaNO3 and NaH2PO4 (0.88 mM NaNO3, 36 &#181;M NaH2PO4</td>
		</tr>
		<tr>
			<td>f/2+-agar</td>
			<td colspan="3">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</td>
		</tr>
		<tr>
			<td>f/2-agar</td>
			<td colspan="3">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 &#181;M NaH2PO4</td>
		</tr>
		<tr>
			<td>Trace element solution</td>
			<td colspan="3">0.36 mM NaH2PO4, 12 &#181;M Na2EDTA, 39 nM CuSO4, 26 nM Na2MoO4 , 77 nM ZnSO4, 42 nM CoCl2, 0.91 &#181;M MnCl2</td>
		</tr>
		<tr>
			<td>Vitamin solution</td>
			<td colspan="2">0.3 &#181;M thiamin-HCl, 2.1 nM biotin, 0.37 nM cyanocobalamin</td>
			<td></td>
		</tr>
	</tbody>
</table>

natural transformation, protein expression, and cryoconservation of the filamentous cyanobacterium phormidium lacuna

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。

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?...

Watch this Scientific Journal Video about Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna at JoVE.com

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

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。

丝状蓝藻腔隙的自然转化、蛋白表达和低温保存

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

该协议描述了属于振荡器的丝状蓝藻如何通过自然转化进行转化。它还展示了如何研究不同类型的运动。自然变换是最简单的变换技术，如果它有效的话。只需要DNA，细胞和生长培养基。到目前为止，没有其他振荡器成员可以通过自然变换来转化。我们希望我们的研究能刺激其他小组尝试其他振荡器。对于转化，要进行良好，健康的外观培养和良好的DNA制备。对于运动研究，始终使用超声处理培养物。治疗应新鲜。演示该程序的将是来自我实验室的技术人员Nora Weber。首先将50毫升液体F2培养基接种到两个250毫升烧瓶中，每瓶来自运行培养物的一毫升P.lacuna细丝。在25摄氏度的激越下在白光下培养约五天。五天后，以10， 000 RPM匀浆100毫升P.lacuna细胞悬浮液三分钟，并在750纳米处测量光密度。然后，以6， 000倍G离心细胞悬浮液15分钟，除去上清液并将沉淀悬浮在800微升剩余液体和额外的F2 +培养基中。取八个F2 + Bacto琼脂平板，每毫升含有120微克卡那霉素，并将10微克DNA移液到每个琼脂平板的中间。立即移液100微升的细胞悬浮液在DNA的顶部。将琼脂板放在干净的工作台上，不要盖上盖子，以使多余的液体蒸发。关闭盘子，在25摄氏度的白光下培养两天。两天后，将每个琼脂平板的细丝分配到几个新鲜的F2 + Bacto琼脂平板上，每毫升卡那霉素含有120微克，并带有接种环。在25摄氏度的白光下培养板，并在显微镜下定期检查培养物。14至28天后，识别死亡的褐色细丝，并在显微镜下寻找转化的细丝。转化后的细丝看起来健康和绿色，与大部分细丝不同。将每个转化后的长丝转移到50毫升的烧瓶中，其中10毫升F2 +培养基，每毫升卡那霉素250微克。在25摄氏度的白光下摇床上培养，观察生长长达四周。将细丝转移回含有每毫升卡那霉素250微克的琼脂培养基中，并等待细丝生长。然后，再次增加卡那霉素浓度以加速分离。对于GFP表达，用荧光显微镜以40X或63X放大倍率观察单丝，并捕获明场透射图像和荧光图像。在50 RPM水平搅拌下在白光下在F2培养基中培养P.voida约五天，直到750纳米处的估计光密度变为0.35。将样品储存在四摄氏度。用超声波以最大功率和循环使细丝均质一分钟。测量750纳米的光密度，并将含有P.lacuna的8毫升介质转移到六厘米的培养皿中。等待几分钟，直到样品达到室温，然后用玻璃纸箔覆盖培养皿。将显微镜载玻片放在带相机的标准显微镜的X-Y台上。打开显微镜灯，将4X或10X物镜移动到光的路径中。然后，将培养皿放在幻灯片的顶部。通过工作台的 X、Y 和 Z 运动调整单细丝或灯丝束。观察单丝或束的运动，并使用标准显微镜相机记录运动。确保物镜不接触液体。移液0.5毫升含有P.lacuna的溶液在六厘米培养皿的Bacto琼脂表面上。让液体进入表面。大约20分钟后，关闭培养皿，并使用4X或10X物镜观察细丝在表面上的运动。使用目测摄像头和微型计算机系统捕捉延时记录。细丝必须首先通过眼睛聚焦，然后通过眼部摄像头。确保后续图像之间的时间间隔为五秒到一分钟。对微型计算机的Linux脚本进行编程，以控制延时记录。准备安装五毫米LED的LED支架，从下面到上方照射20毫米正方形的区域。测量和调整 LED 强度。确保整个设置位于黑暗的房间或封闭的黑暗容器中。将八毫升含有P.lacuna的培养基放入六厘米的培养皿中，用盖子关闭培养皿，并将其放在LED支架上，以使LED位于培养皿的中心。通常在两天后，用智能手机摄像头直接瞄准光处理位置捕获培养皿的图像。使用支架和白色纸张作为背景，以确保每张照片的距离和光线条件相同。打开ImageJ软件，单击“文件”，“打开”，然后选择文件。然后，单击 Enter。选择笔直按钮，然后按鼠标左键，从培养皿的一端到另一端画一条线。确保线穿过细丝圆的中心。单击“分析和测量”以查看培养皿的长度。然后，单击“图像J”菜单中的“分析和绘制配置文件”。估计圆圈外像素强度的平均值和圆圈内像素强度的另一个平均值。将鼠标指向这些值之间的 Y 位置，以估计圆两侧的 X 值。记下这两个值并计算差值。然后选择直线按钮，并在像素强度的中间最大值处绘制一条线。最后，单击“分析”，然后单击“测量”以获取内细胞圆圈的长度。此处显示了P.lacuna与PAK1转化后插入物的整合和分离。在转化后一周左右使用外部引物进行的PCR测试通常在电泳凝胶上有两条条带，一条具有野生型条带的大小，另一条较慢的迁移条带表明电阻盒的插入。此处，泳道 1 至 4 表示在分离耐药丝 7 天、11 天、14 天和 17 天后丝的 PCR 产物，泳道 5 表示野生型 PCR 产物。在七天的样品中，插入片段存在于染色体的一小部分中。该分数增加至17天，其中没有野生型条带可见;也就是说，分离是完全的。该图像代表在内源性藻蓝蛋白β启动子控制下sfGHP表达的载体pMH1。此处显示了 P.lacuna 同源序列、pUC19 载体主链以及具有 sfGFP 和卡那霉素耐药性的插入片段。在pMH1中，sfGHP基因被置于藻蓝蛋白β基因的三个素数中，因此由内源性cpcβ启动子驱动。此处显示了P.lacuna野生型细丝以及与PAK1，PAK2，PAK3和pMH1转化后的荧光图像。sfGFP的表达由cpc 560启动子，a2813启动子，psbA2S启动子或内源性cpcβ启动子驱动。这里展示的合并图像显示了Phormidium lacuna的运动性。琼脂表面的运动如图所示。时间间隔为一分钟。液体介质中的运动在这里介绍。时间间隔为 10 秒。自然变换是一种简单的方法。只需将质粒DNA与具有同源序列的抗性盒与细胞混合，然后让细胞在含有抗生素的培养基上生长。基因可以被灭活，蛋白质可以被过度表达。这对于任何基础研究和生物技术研究都很重要。在也建立了自然转化的单细胞蓝藻中，讨论了光合作用或光感受器等的分子研究。

Research

JoVE Journal

Biology

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::ÃƒÅ½Ã‚Â²-glucuronidase Reporter Gene Expression

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...