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Calculation of Microorganism Lag Times as a Measure of Adaptative Capability between Different Growth Conditions
采用微生物滞后时间计算衡量不同生长条件下的自适应能力

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Abstract

This protocol has been designed as a simple and efficient way to investigate microorganism adaptive capabilities (Enjalbert et al., 2015). It is performed using switch experiments in which cells are initially grown in in the first condition (primary cultures), then rapidly switched to the second condition (secondary culture) without centrifugation or quenching. The measurement is based on the capacity of the secondary culture cells to resume growth. This protocol can be utilized for assessing metabolic or stress adaptation of microorganisms.

Keywords: Lag(滞后), Switch(开关), Growth(生长), Adaptation(适应)

Materials and Reagents

  1. Sterile Erlen-Meyer flask per switch
  2. 0.45 µm filter per switch (Minisart 0.45 μm filter) (Sartorius AG)
  3. Plastic adapter per switch (Tube versilic 4 x 7 mm) (Saint-Gobain or equivalent)
  4. Sterile syringe 5 ml (Terumo Medical Corporation)
  5. Microorganism culture in condition 1
  6. Medium for growth condition 2

Equipment

  1. Shaker incubator
  2. Spectrophotometer and cuvettes

Procedure


Figure 1. Switch experiments procedure

  1. In the incubator, pre-warm one empty 5 ml syringe, two 5 ml syringes filled with 3 ml condition 2 medium, the flask filled with 30 ml of condition 2 medium, and the filter with a plastic adapter on the nozzle. Sterility of the media has to be maintained at this step.
  2. With the empty syringe (needle inner diameter: 0.8 mm), sample cells from the primary culture in condition 1.
    Notes:
    1. The volume depends on two factors. (i) It has to be maximized so that the absorbance when eluted in the condition 2 flask can be reproducibly measured at step 6 (for example, 2 ml of E. coli culture at OD600 nm = 3 in the primary culture provide a measurable OD600 nm = 0.2 in the secondary culture). (ii) It has to be minimized to avoid clogging the filter at step 3.
    2. From step 2 onward, the manipulations have to be performed as rapidly as possible to minimize the culture perturbation (i.e. in less than 2 min). As a control, we suggest performing the same experiment by replacing condition 2 medium by condition 1 and ensuring that the initial growth rate in the secondary culture is equal to the growth rate in the primary culture.
  3. Attach the filter to syringe filled with 3 ml of pre-warmed condition 2 medium and rinse the cells on filter (the flow-through is discarded).
  4. Replace the syringe by one of the two syringes filled with 3 ml of pre-warmed condition 2 medium and rinse the cells on the filter with medium 2.
  5. Attach the second 5 ml syringe filled with 3 ml of pre-warmed condition 2 medium on the other end of the filter using the plastic adapter.
  6. Elute the cells over the flask filled with 30 ml of condition 2 medium.
  7. Place the flask in the shaker incubator for 1 min to ensure homogeneity of the solution and measure the initial OD. Immediately place the flask back in the incubator.
  8. Monitor again the growth of the secondary culture when the cells reach their maximal growth rate on condition 2 (standardized by trial). For example, switching E. coli from a glucose to an acetate based mineral medium required measuring the OD at times 60 and 90 minutes after inoculation by spectrophotometry at OD600 nm (Enjalbert et al., 2015). The measure at 60 min allows to calculate the lag (see step 8), and the calculation of the growth rate between 60 and 90 min ensures that the cells reach their maximal growth rate (this maximum growth rate in condition 2 has to be previously determined).
  9. Use the following equation to calculate the lag time before maximal growth (see Figure 2 for justification):
    (t1-tm) = (t1-t0)-ln(X1/X0)/μmax


    Figure 2. Theoretical growth profiles for the calculation of the lag in the switch experiments. tm is the theoretical time needed to increase the biomass from X0 to X1 if the growth rate is maximal (µmax) from t0. If there is a delay, X1 will be obtained at t1 (i.e. later than tm). From these elements, the lag can be determined as (t1-tm).

    By definition,
    Eq1: μmax = ln(X1/X0)/(tm-t0)
    From which
    Eq2: (tm-t0)= ln(X1/X0)/μmax
    Since
    Eq3: (t1-tm)= (t1-t0)-(tm-t0)
    From Eq3 and Eq2,
    Eq41: (t1-tm) = (t1-t0)-ln(X1/X0)/μmax

Acknowledgments

B. E. chair was supported by the INRA (Institut National de la Recherche Agronomique) and the INSA (Institut National des Sciences Appliquées) (Program <Chaire d’excellence>). We thank Pierre Millard, Alessandra Fontana and Andrea Belluati for their worthy contributions.

References

  1. Enjalbert, B., Cocaign-Bousquet, M., Portais, J. C. and Letisse, F. (2015). Acetate exposure determines the diauxic behavior of Escherichia coli during the glucose-acetate transition. J Bacteriol 197(19): 3173-3181.

简介

该协议被设计为一种简单有效的方法来研究微生物适应能力(Enjalbert等人,2015)。 其使用开关实验进行,其中细胞最初在第一条件(原代培养物)中生长,然后快速切换至第二条件(二次培养)而不进行离心或猝灭。 测量基于次级培养细胞恢复生长的能力。 该方案可用于评估微生物的代谢或胁迫适应。

关键字:滞后, 开关, 生长, 适应

材料和试剂

  1. 每个开关灭菌Erlen-Meyer瓶
  2. 每个开关0.45μm滤光器(Minisart0.45μm滤光器)(Sartorius AG)
  3. 每个开关塑料适配器(Tube versilic 4 x 7 mm)(Saint-Gobain或同等产品)
  4. 无菌注射器5ml(Terumo Medical Corporation)
  5. 条件1的微生物培养
  6. 生长条件2的培养基

设备

  1. 摇床孵化器
  2. 分光光度计和比色皿

程序


图1.切换实验过程

  1. 在孵化器中,预热一个空的5ml注射器,两个5ml注射器充满3ml条件2培养基,烧瓶装有30ml条件2培养基,以及在喷嘴上带有塑料适配器的过滤器。在该步骤中必须保持介质的无菌性。
  2. 用空的注射器(针内径:0.8mm),在条件1下来自原代培养物的样品细胞 注意:
    1. 音量取决于两个因素。 (i)必须使其最大化 在条件2烧瓶中洗脱时的吸光度可以重复 在步骤6测量(例如,在OD 600nm = 3时2ml大肠杆菌培养物 ?在原代培养物中提供可测量的OD 600nmλ= 0.2 继代培养)。 (ii)必须尽量减少,以避免堵塞 在第3步过滤。
    2. 从步骤2开始,操作必须 尽可能快地执行以使培养物摄动最小化 ?(即在小于2分钟内)。作为一个控制,我们建议执行相同 ?通过用条件1代替条件2培养基并确保实验 在次级培养中的初始生长速率等于 原代培养物的生长速率。
  3. 将过滤器连接到填充有3ml预热条件2培养基的注射器,并冲洗过滤器上的细胞(流出物被丢弃)。
  4. 用装有3 ml预热条件2培养基的两个注射器之一更换注射器,并用培养基2冲洗过滤器上的细胞。
  5. 使用塑料适配器将填充有3ml预热条件2培养基的第二个5 ml注射器连接到过滤器的另一端。
  6. 将细胞洗脱在装有30ml条件2培养基的烧瓶上。
  7. 将烧瓶置于摇床培养箱中1分钟,以确保溶液的均匀性,并测量初始OD。立即将烧瓶放回培养箱。
  8. 当条件2(通过试验标准化)细胞达到其最大生长速率时,再次监测次级培养物的生长。例如,切换 E。大肠杆菌从葡萄糖到基于乙酸盐的矿物质培养基中培养,需要在OD 600nm处通过分光光度法接种后60和90分钟时测量OD(Enjalbert et al。 >,2015)。在60分钟的测量允许计算滞后(参见步骤8),并且在60和90分钟之间的生长速率的计算确保细胞达到其最大生长速率(条件2中的该最大生长速率必须预先确定)。
  9. 使用以下公式计算最大生长前的滞后时间(参见图2的对齐):
    (t sub-t m)=(t 1 -t -t 0)-In(X sub-1) 1 /X 0 )/μ max


    图2.用于计算切换实验中的滞后的理论生长曲线 t是从X 0增加生物质所需的理论时间,如果生长速率从t 0开始是最大的(μ),则将其设置为X <1>。如果存在延迟,则将在t sub1(,即晚于t sub )获得X sub1 。从这些元素,滞后可以被确定为(t 1 -t -t m)。

    按照定义,
    公式1:μsub max = sub(X sub/X sub)/(t sub) 0 )
    从中
    等式2:(t sub-t sub)= ln(X sub/X sub)/μ max
    因为
    方程3:(t 1 -t 1 m)=(t 1 -t 1 0) - (t m -t 0
    从Eq3和Eq2,
    方程41:(t 1 -t 1 m)=(t 1 -t 1 0) - ln(X 1) sub> 1 /X 0 )/μ max

致谢

BE椅子由INRA(国立农业研究所)和INSA(国立科学研究所应用物品研究所)( chaire d'excellence > )。我们感谢皮埃尔·米勒德,亚历山德拉·丰塔纳和安德烈·贝卢拉提他们的价值贡献。

参考文献

  1. Enjalbert,B.,Cocaign-Bousquet,M.,Portais,J.C.and Letisse,F。(2015)。 乙酸暴露决定了大肠杆菌在葡萄糖醋酸盐中的二氢化作用transition。 197(19):3173-3181。
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引用:Enjalbert, B. (2016). Calculation of Microorganism Lag Times as a Measure of Adaptative Capability between Different Growth Conditions . Bio-protocol 6(3): e1727. DOI: 10.21769/BioProtoc.1727.
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