搜索

Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters
肠杆菌科中受阿拉伯糖调节启动子调控的基因剂量实验   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli cell expressing this protein.

Keywords: Gene-dosage toxicity(基因剂量毒性), Arabinose(阿拉伯糖), Protein abundance(蛋白质丰度), Western-blot(免疫印迹), Fitness(适应度), Over-expression(过表达), Plasmid(质粒)

Background

Gene dosage experiments are crucial for understanding the effects of protein over-expression on fitness and determining the optimal levels of protein abundance. Several genes are toxic even when expressed at very low levels. It is therefore important to express the protein from a tightly regulated promoter to minimize leaky expression. Here we have elucidated conditions for expression of proteins under the well-characterized arabinose induced pBAD promoter, and designed protocols to measure the intracellular abundance and fitness of E. coli cells harboring the overexpression plasmid.

Materials and Reagents

  1. Sterile tips
  2. 1.5 ml microfuge tubes (Corning, Axygen®, catalog number: MCT-175-C )
  3. 14 ml polypropylene tubes for bacterial culture (Corning, Falcon®, catalog number: 352059 )
  4. 50 ml tubes for bacterial culture (Corning, Falcon®, catalog number: 352070 )
  5. Sterile needle for inoculation (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 253988 )
  6. Honeycomb plates (Bioscreen, catalog number: 95025BIO )
  7. Gene of interest cloned in pBAD/MCS-vector (https://www.embl.de/pepcore/pepcore_services/strains_vectors/vectors/bacterial_expression_vectors/popup_bacterial_expression_vectors/ obtained from European Molecular Biology Laboratory EMBL) using appropriate restriction sites in the multiple cloning site
  8. E. coli BW27783 cells (CGSC, catalog number: 12119 )
  9. LB/Agar plates
  10. Ampicillin (Sigma-Aldrich, catalog number: A0166 )
  11. Sodium phosphate buffer, pH 7.4
  12. Tris-HCl pH 8.0
  13. Pierce BCA protein assay kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23227 )
  14. 4x protein gel loading dye (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0007 )
  15. Pre-cast 12% Bis-Tris SDS polyacrylamide gel (Bio-Rad Laboratories, catalog number: 3450123 )
  16. Trans-blot Turbo midi nitrocellulose transfer packs (Bio-Rad Laboratories, catalog number: 1704158 )
  17. Antibody raised against the protein of interest
  18. Western-breeze Chromogenic Immunodetection kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: WB7105 for anti-rabbit)
  19. M9 salts (BD, DifcoTM, catalog number: 248510 )
  20. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
  21. Thiamine (Sigma-Aldrich, catalog number: T1270 )
  22. Casamino acid (AMRESCO, catalog number: J851 )
  23. Glucose (Sigma-Aldrich, catalog number: G7021 )
  24. L(+) arabinose (EMD Millipore, catalog number: 178680 )
  25. 10x Bugbuster reagent (Novagen, catalog number: 70921-3 )
  26. Benzonase nuclease (EMD Millipore, catalog number: 70664 )
  27. Supplemented M9 medium (see Recipes)
  28. Lysis buffer (see Recipes)
  29. L(+) arabinose solution (Concentrations used) (see Recipes)

Equipment

  1. Pipettes
  2. Incubator
  3. Heated Orbital shaker
  4. Centrifuge (Eppendorf, models: 5810 R and 5417 R )
  5. Spectrophotometer (BioTek Instruments, model: PowerWave HT )
  6. Rotator-mixer
  7. Trans-Blot Turbo transfer system (Bio-Rad Laboratories, catalog number: 1704155 )
  8. Bioscreen C (Growth Curves USA)

Software

  1. ImageJ software

Procedure

  1. For abundance measurement
    1. Transform pBAD plasmid containing the gene of interest in to electro-competent BW27783 cells (see Notes, point #1), and spread on LB/Agar plates containing 100 µg/ml ampicillin. Incubate plates overnight at 37 °C.
    2. Next day, pick a single colony from the plate and inoculate into 2 ml of supplemented M9 medium (see Recipes and Notes, point #2) containing 100 µg/ml of ampicillin. Grow overnight with shaking (250 rpm) at 37 °C in 14 ml polypropylene tubes.
    3. Next day, dilute 500 µl of the overnight culture 1/100 in to 50 ml of fresh M9 medium containing 100 µg/ml of ampicillin and varying concentrations of arabinose (0-0.05%) (see Recipes), and grow for 4 h with shaking (250 rpm) at 37 °C.
    4. After 4 h, measure OD600 of the cultures, and spin down in a table-top centrifuge at 3,000 x g for 15 min. Aspirate as much culture supernatant as possible. Store the cell pellets at -20 °C.
    5. Prepare the lysis buffer, which is the buffer of choice (50 mM sodium phosphate buffer, pH 7.4, 10 mM Tris-HCl pH 8.0, etc.) supplemented with the detergent Bugbuster and Benzonase nuclease (see Recipes below). Based on the measured OD600 of the cultures, re-suspend the pellets in the prepared lysis buffer such that the final OD600 is 2.0 (see Notes, point #3). Allow the lysis to proceed for 20 min at room temperature on a rotator-mixer.
    6. Following lysis, spin down the cell debris for 30 min at 7500 x g in a centrifuge that has been pre-chilled at 4 °C.
    7. Separate the supernatant and quantify the total protein in each sample using the BCA assay kit using the manufacturer’s instructions.
    8. Load 20 µl of the supernatants on a 12% Bis-Tris SDS polyacrylamide gel, after required dilution of the samples (see Notes, point #4).
    9. Resolve the samples at a voltage gradient of 10 V/cm of gel.
    10. Once the dye front has reached the base, the gel is ready to be transferred to a membrane for blotting. Use pre-assembled nitrocellulose membrane sandwiches (see Notes, point #5).
    11. Following transfer, wash the membrane with water two times to remove transfer buffer components and weakly bound proteins. From this step onwards, use Invitrogen’s Western-breeze Chromogenic Immunodetection kit to develop the membrane.

  2. For growth rate measurement
    1. From step A2 above, dilute the overnight culture to a final OD600 of 0.01 (see Notes, point #3) into fresh M9 medium containing 100 µg/ml of ampicillin and varying concentrations of arabinose (see Recipes). Aliquot 150 µl of this into three wells of the honeycomb Bioscreen plate (see Notes, point #6). This serves as replicates for a single colony. To obtain standard error of biological replicates, inoculate 3 independent colonies, and subject each of them to varying arabinose concentration.
    2. Aliquot 150 µl M9 medium into three independent wells. This serves as the background for OD measurements.
    3. Measure OD600 values at 15 min intervals over a period of 12 h in Bioscreen C system at 37 °C with shaking (see Notes, point #6).

Data analysis

  1. Use ImageJ software to evaluate the band intensities from the Western blot (For a complete tutorial, please visit ‘https://imagej.nih.gov/ij/docs/guide/146-30.html#toc-Subsection-30.13’).
  2. Normalize the intensity by the total protein concentration obtained by BCA assay, and also scale up the values by the dilution factor. If the protein of interest is an endogenous E. coli protein, then calculate the fold-overexpression of the protein of interest based on the intensity obtained for untransformed BW27783 cells (set to 1). For a foreign protein, the expression level obtained with 0% arabinose should be set to 1.
  3. Fit the growth curves obtained from Bioscreen C as vs. time using the following 4-parameter Gompertz equation to obtain growth rate parameters (Adkar et al., 2017).



    Where,  is the fold-increase over initial population at saturation,  is the shape factor and defined as  where  is the maximum growth rate, and the lag time is the time taken to achieve the maximum growth rate.
  4. Make a plot of gene-dosage effect using measured growth rates and intracellular protein abundance (Figure 1) (see Notes, point #7).


    Figure 1. Representative gene-dosage toxicity curves for E. coli Dihydrofolate Reductase (DHFR) expressed from pBAD-plasmid (Bhattacharyya et al., 2016). A. Plot of relative growth rate as a function of arabinose concentration; B. Plot of relative growth rate as a function of intracellular abundance of DHFR protein.

Notes

  1. The transporter araE in E. coli helps in uptake of arabinose from the medium. However, as the expression of araE is all or none in WT E. coli cells (e.g., MG1655), induction with arabinose results in a heterogeneous population. BW27783 that is used in this protocol is a strain of E. coli MG1655 that has been engineered to constitutively express araE, resulting in a uniform and homogeneous uptake of arabinose (Khlebnikov et al., 2001).
  2. The gene-dosage experiments can be done in any medium of choice (LB, M9, etc.). However over-expression of the protein of interest may show different phenotypes in different media conditions. For example as discussed in (Bhattacharyya et al., 2016), over-expression of E. coli Dihydrofolate Reductase (DHFR) was found to be toxic in supplemented M9 medium, but not in LB medium. We therefore suggest choosing the growth medium which allows the study of the phenotype of interest.
  3. OD600 is defined for 1 cm path-length.
  4. The typical amount of total protein from cell lysate loaded on to the gel was 5 μg. However, for higher levels of induction, dilution will be necessary. We therefore suggest doing a pilot experiment to find the necessary dilution.
  5. Instead of pre-assembled nitrocellulose membrane sandwiches, one can prepare their own using pre-cut nitrocellulose membranes, blotting papers, and tris-glycine-methanol transfer buffer to perform this step successfully.
  6. Bioscreen C is an absorbance based microplate reader that is used to measure growth curves of microorganisms (http://www.growthcurvesusa.com/description.html). As opposed to a conventional microplate reader, which can measure 96 wells at a time, honeycomb plates used in Bioscreen C have 100 wells, and two plates can be used at a time. The design of the honey comb plate ensures uniform temperature across the wells without any significant condensation/evaporation, thereby greatly reducing errors among replicates. However, it should be mentioned that in absence of Bioscreen C instrument, any conventional microplate reader or spectrophotometer with cuvette can also be used to perform this step successfully.
  7. If it is desirable to find out the absolute amount of protein required to achieve a particular growth inhibition, it can be done by purifying the protein of interest, estimating its concentration and then using a known amount of the purified protein as a standard/reference to estimate the amount of protein from the crude lysate at each given induction level.

Recipes

  1. Supplemented M9 medium (1 L)
    11.28 g of M9 salts (Difco) (identical to the classical M9 salts recipe in Molecular Cloning by Maniatis)
    1 ml 1 M MgSO4
    5 µl 100 mM thiamine
    10 ml 10% casamino acid
    10 ml 20% glucose
    Make up the volume to 1,000 ml, sterilize by filtration
  2. Lysis buffer (10 ml)
    1 ml 10x Bugbuster reagent
    10 µl Benzonase nuclease
    Make up the volume to 10 ml using buffer of choice
  3. L(+) arabinose solution (concentrations used)
    Start with 0.05%, and then do 4-fold serial dilution up to 1.22 x 10-5%, in addition to 0% arabinose

Acknowledgments

This work was funded by NIH RO1 GM111955.

References

  1. Adkar, B. V., Manhart, M., Bhattacharyya, S., Tian, J., Musharbash, M. and Shakhnovich, E. I. (2017). Optimization of lag phase shapes the evolution of a bacterial enzyme. Nature Ecology & Evolution 1: 0149.
  2. Bhattacharyya, S., Bershtein, S., Yan, J., Argun, T., Gilson, A. I., Trauger, S. A. and Shakhnovich, E. I. (2016). Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity. Elife 5.
  3. Khlebnikov, A., Datsenko, K. A., Skaug, T., Wanner, B. L. and Keasling, J. D. (2001). Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology 147(Pt 12): 3241-3247.

简介

该方案用于测定蛋白质过表达对E适应度的影响。大肠杆菌。 它基于来自阿拉伯糖调节的pBAD启动子的目的蛋白的质粒表达,随后通过Western印迹测量细胞内蛋白质丰度以及E的生长参数的测量。 表达该蛋白质的大肠杆菌细胞。
【背景】基因剂量实验对于了解蛋白质过表达对健康的影响和确定蛋白质丰度的最佳水平至关重要。 几个基因即使在非常低的水平表达也是有毒的。 因此,重要的是从紧密调节的启动子表达蛋白质以使渗漏表达最小化。 在这里,我们已经阐明了在良好表征的阿拉伯糖诱导的pBAD启动子下的蛋白质表达的条件,并且设计了用于测量E细胞内丰度和适应度的方案。 含有过表达质粒的大肠杆菌细胞。

关键字:基因剂量毒性, 阿拉伯糖, 蛋白质丰度, 免疫印迹, 适应度, 过表达, 质粒

材料和试剂

  1. 无菌提示
  2. 1.5ml微量离心管(Corning,Axygen ,目录号:MCT-175-C)
  3. 14ml用于细菌培养的聚丙烯管(Corning,Falcon ®,目录号:352059)
  4. 50ml细菌培养管(Corning,Falcon ®,目录号:352070)
  5. 用于接种的无菌针(Thermo Fisher Scientific,Thermo Scientific TM,目录号:253988)
  6. 蜂窝板(Bioscreen,目录号:95025BIO)
  7. 克隆在pBAD / MCS载体中的感兴趣的基因( https://www.embl.de/pepcore/pepcore_services/strains_vectors/vectors/bacterial_expression_vectors/popup_bacterial_expression_vectors/
  8. 电子。大肠杆菌 BW27783细胞(CGSC,目录号:12119)
  9. LB /琼脂板
  10. 氨苄青霉素(Sigma-Aldrich,目录号:A0166)
  11. 磷酸钠缓冲液,pH 7.4
  12. Tris-HCl pH 8.0
  13. Pierce BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23227)
  14. 4x蛋白凝胶加载染料(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0007)
  15. 预制的12%Bis-Tris SDS聚丙烯酰胺凝胶(Bio-Rad Laboratories,目录号:3450123)
  16. Trans-blot Turbo midi硝酸纤维素转移包(Bio-Rad Laboratories,目录号:1704158)
  17. 针对感兴趣的蛋白质产生抗体
  18. Western-breeze显色免疫检测试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:WB7105,用于抗兔)
  19. M9盐(BD,Difco TM,目录号:248510)
  20. 七水硫酸镁(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:230391)
  21. 硫胺素(Sigma-Aldrich,目录号:T1270)
  22. 卡氨酸(AMRESCO,目录号:J851)
  23. 葡萄糖(Sigma-Aldrich,目录号:G7021)
  24. L(+)阿拉伯糖(EMD Millipore,目录号:178680)
  25. 10x Bugbuster试剂(Novagen,目录号:70921-3)
  26. Benzonase核酸酶(EMD Millipore,目录号:70664)
  27. 补充M9培养基(见食谱)
  28. 裂解缓冲液(见配方)
  29. L(+)阿拉伯糖溶液(使用浓度)(参见食谱)

设备

  1. 移液器
  2. 孵化器
  3. 加热轨道摇床
  4. 离心机(Eppendorf,型号:5810 R和5417 R)
  5. 分光光度计(BioTek Instruments,型号:PowerWave HT)
  6. 旋转搅拌机
  7. Trans-Blot Turbo Transfer系统(Bio-Rad Laboratories,目录号:1704155)
  8. Bioscreen C(美国增长曲线)

软件

  1. ImageJ软件

程序

  1. 丰度测量
    1. 将含有感兴趣的基因的pBAD质粒转化到电受感染的BW27783细胞(参见注释,点#1),并在含有100μg/ ml氨苄青霉素的LB /琼脂平板上铺展。 37℃孵育过夜。
    2. 第二天,从板上挑一个菌落,并接种到含有100μg/ ml氨苄青霉素的2ml补充的M9培养基(参见食谱和注释,点号2)中。在37℃下,在14ml聚丙烯管中摇动(250rpm)生长过夜。
    3. 第二天,将500μl的过夜培养物1/100稀释到50ml含有100μg/ ml氨苄青霉素和不同浓度的阿拉伯糖(0-0.05%)(参见食谱)的新鲜M9培养基中,并且生长4小时,在37℃下摇动(250rpm)
    4. 4小时后,测量培养物的OD 600,并以3000×g离心在台式离心机中15分钟。吸出尽可能多的培养上清液。将细胞沉淀物储存在-20°C
    5. 准备裂解缓冲液,该缓冲液是补充有洗涤剂Bugbuster和Benzonase核酸酶的缓冲液(50mM磷酸钠缓冲液,pH7.4,10mM Tris-HCl pH8.0,等等),参见食谱下面)。基于测量的培养物的OD 600,将沉淀重新悬浮在制备的裂解缓冲液中,使得最终OD 600为2.0(参见注释,点#3) 。允许裂解在室温下在旋转器 - 混合器上进行20分钟。
    6. 在裂解后,在已经在4℃预冷的离心机中以7500×g / min旋转细胞碎片30分钟。
    7. 使用BCA测定试剂盒,使用制造商的说明书分离上清液并定量每个样品中的总蛋白质。
    8. 在需要稀释样品后,在12%Bis-Tris SDS聚丙烯酰胺凝胶上加载20μl上清液(见注释,第4点)。
    9. 以10 V / cm的凝胶电压梯度解析样品。
    10. 一旦染料前端已经到达碱,就可以将凝胶转移到膜上进行印迹。使用预先组装的硝酸纤维素膜三明治(见注释,第5点)
    11. 转移后,用水洗两次以除去转移缓冲液组分和弱结合蛋白。从此步骤开始,使用Invitrogen的Western-Breeze Chromogenic Immunodetection试剂盒开发膜
  2. 用于增长率测量
    1. 从上述步骤A2,将过夜培养物稀释至含有100μg/ ml氨苄青霉素和不同浓度阿拉伯糖的新鲜M9培养基中的0.01(参见注释,点#3)的最终OD 600(参见食谱)。将等分试样将其加入到蜂窝Bioscreen板的三个孔中(参见注释,第6点)。这是一个殖民地的复制品。为了获得生物重复的标准误差,接种3个独立的菌落,并对其中的每一个进行变异的阿拉伯糖浓度。
    2. 将150μlM9培养基分成三个独立的孔。这是OD测量的背景。
    3. 在Bioscreen C系统中,在37℃下振荡,在12小时内以15分钟间隔测量OD 600值(参见注释,第6点)。

数据分析

  1. 使用ImageJ软件评估蛋白质印迹中的条带强度(有关完整的教程,请访问“ https:// imagej。 nih.gov/ij/docs/guide/146-30.html#toc-Subsection-30.13 的')。
  2. 通过BCA测定法获得的总蛋白质浓度使强度达到标准化,并且还可以通过稀释因子来扩大值。如果感兴趣的蛋白质是内源性蛋白质。大肠杆菌蛋白质,然后基于未转化的BW27783细胞获得的强度(设置为1)计算目的蛋白质的倍数过表达。对于外来蛋白质,用0%阿拉伯糖获得的表达水平应设定为1.
  3. 将从Bioscreen C获得的生长曲线拟合为 vs.  time 使用以下4参数Gompertz方程来获得生长速率参数(Adkar等人,2017)。



    其中,是饱和时初始人口的倍数增加, img width =“10”height =“11”alt =“”src =“/ attached / image / 20170712 / 20170712182008_5900.jpg”/>是形状因子,并定义为其中  是最大增长率,滞后时间是时间取得最大增长率。
  4. 使用测量的生长速率和细胞内蛋白质丰度来绘制基因剂量效应(图1)(见注释,第7点)。


    图1.代表性基因剂量毒性曲线 E。从pBAD质粒表达的二氢叶酸还原酶(DHFR)(Bhattacharyya等人,2016)。 A.作为阿拉伯糖浓度的函数的相对生长速率曲线图B.相对生长率作为DHFR蛋白质细胞内丰度的函数图

笔记

  1. E中的运输商araE。大肠杆菌有助于从培养基中摄取阿拉伯糖。然而,由于araE的表达在WT E中是全部的或没有的。大肠杆菌细胞(例如,,MG1655),用阿拉伯糖诱导导致异质群体。本协议中使用的BW27783是一种E型菌株。大肠杆菌MG1655已经被设计成组成型表达araE,导致阿拉伯糖的均匀和均匀摄取(Khlebnikov等人,2001)。
  2. 基因剂量实验可以在任何选择的培养基(LB,M9,等等)中完成。然而,感兴趣的蛋白质的过度表达可以在不同的培养基条件下显示不同的表型。例如,(Bhattacharyya等人,2016)所讨论的,E,E的过度表达。发现二氢叶酸还原酶(DHFR)在补充的M9培养基中是有毒的,但不在LB培养基中。因此,我们建议选择允许研究感兴趣的表型的生长培养基。
  3. OD 600 被定义为1厘米的路径长度。
  4. 加载到凝胶上的细胞裂解物的总蛋白质的典型量为5μg。然而,对于较高的诱导水平,稀释是必要的。因此,我们建议进行试点实验,以找出必要的稀释度
  5. 而不是预先组装的硝酸纤维素膜三明治,可以使用预先切割的硝酸纤维素膜,吸墨纸和三甘氨酸 - 甲醇转移缓冲液来制备自己的成功执行此步骤。
  6. Bioscreen C是用于测量微生物生长曲线的基于吸光度的酶标仪( http://www.growthcurvesusa.com/description.html )。与传统的微孔板读数器相比,可以一次测量96孔,Bioscreen C中使用的蜂窝板有100个孔,一次可以使用两个板。蜂巢梳板的设计确保井间温度均匀,无任何明显的冷凝/蒸发,从而大大减少了重复的误差。然而,应该提到的是,在没有Bioscreen C仪器的情况下,任何常规的酶标仪或分光光度计都可以用比色杯来成功执行。
  7. 如果需要找出实现特定生长抑制所需的蛋白质的绝对量,则可以通过纯化目标蛋白质,估计其浓度,然后使用已知量的纯化蛋白质作为标准/参考来进行在每个给定的诱导水平估计来自粗裂解物的蛋白质的量。

食谱

  1. 补充M9培养基(1升)
    11.28g M9盐(Difco)(与Maniatis分子克隆中的经典M9盐配方相同)
    1ml 1M MgSO 4
    5μl100 mM硫胺素
    10ml 10%酪蛋白氨基酸
    10 ml葡萄糖20% 将体积补充至1000毫升,通过过滤灭菌
  2. 裂解缓冲液(10ml)
    1 ml 10x Bugbuster试剂
    10μlBenzonase nuclease
    使用选择的缓冲区
    将体积调整为10 ml
  3. L(+)阿拉伯糖溶液(使用浓度)
    以0.05%开始,然后进行4倍连续稀释至1.22×10 -5 -5%,除0%阿拉伯糖外,

致谢

这项工作由NIH RO1 GM111955资助。

参考

  1. Adkar,BV,Manhart,M.,Bhattacharyya,S.,Tian,J.,Musharbash,M.and Shakhnovich,EI(2017)。< a class =“ke-insertfile”href =“http:// biorxiv .org / content / early / 2016/11/16/088013“target =”_ blank“>滞后相位的优化塑造了细菌酶的进化。进化 1:0149.
  2. Bhattacharyya,S.,Bershtein,S.,Yan,J.,Argun,T.,Gilson,AI,Trauger,SA和Shakhnovich,EI(2016)。< a class =“ke-insertfile”href =“https ://elifesciences.org/articles/20309“target =”_ blank“>瞬时蛋白质 - 蛋白质相互作用扰乱大肠杆菌代谢组,造成基因剂量毒性。 em> 5.
  3. Khlebnikov,A.,Datsenko,KA,Skaug,T.,Wanner,BL和Keasling,JD(2001)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih通过低亲和力大容量AraE转运蛋白的组成型表达,大肠杆菌中P(BAD)启动子的均一表达。微生物学 147(Pt 12):3241-3247。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Bhattacharyya et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bhattacharyya, S., Bershtein, S. and Shakhnovich, E. (2017). Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters. Bio-protocol 7(14): e2396. DOI: 10.21769/BioProtoc.2396.
  2. Bhattacharyya, S., Bershtein, S., Yan, J., Argun, T., Gilson, A. I., Trauger, S. A. and Shakhnovich, E. I. (2016). Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity. Elife 5.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。