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Protocol for Biotin Bioassay-based Cross Feeding
基于生物测定的生物素交叉喂养法   

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Abstract

Biotin bioassay-based cross-feeding experiments were performed to elucidate the effect on biotin production by bioRbme expression in Agrobacterium tumefaciens (A. tumefaciens) (Feng et al., 2013). The indicator strain used here is the biotin auxotrophic strain of Escherichia coli (E. coli), ER90 (ΔbioF bioC bioD), which was cross-fed by A. tumefaciens species (Feng et al., 2013a). The biotin-free M9 minimal medium plates were formulated as described by other and our research groups (Feng et al., 2013b; Lin et al., 2010; del et al., 1979). Of note, 0.01% (w/v) the redox indicator 2, 3, 5-triphenyl tetrazolium chloride (TTC) was supplemented into the above media. Consequently, biotin generation/production was observed via the reduction of TTC to the insoluble red formazan which is due to the ER90 growth fed by A. tumefaciens strains (Feng et al., 2014). Detailed procedures are described as follows.

Materials and Reagents

  1. Four A. tumefaciens strains [NTL4 (WT), FYJ283 (ΔbioBFDA), FYJ212 (ΔbioRat) and FYJ341 (ΔbioR::Km+bioRbme)]
  2. Biotin auxotrophic strain of E. coli, ER90
  3. 1 nM biotin (Sigma-Aldrich, catalog number: B4510 )
  4. 0.01% (w/v) 2, 3, 5-triphenyl tetrazolium chloride (TTC) (AMRESCO, catalog number: 0 765 )
  5. 0.1% vitamin-free casamino acids hydrolysate (Sigma-Aldrich, catalog number: C7710 )
  6. MgSO4
  7. Glucose
  8. M9 minimal medium (see Recipes)

Equipment

  1. Centrifuge
  2. Petri dishes Petri dishes (90 mm) (Thermo Fisher Scientific, catalog number: 502VF )
  3. Sterile paper disks (6 mm, BBL)

Procedure

  1. Preparation of biotin bioassay plates
    1. The biotin assay plates were prepared as previously described with few changes, one of which referred to the thinner thickness of the plate agar where we dropped paper discs (Feng et al., 2013b; Lin et al., 2010).  
    2. Overnight cultures of strain ER90 (grown in 6 ml of defined M9 minimal medium with 1 nM biotin at 30 °C) were collected by centrifugation (3,600 rpm, 16 min), washed three times with the same volume (6 ml) of M9 medium, and were cultivated at 37 °C to 0.8 OD600 in 200 ml of M9 minimal medium containing 1 nM biotin.
    3. To remove excess of biotin, all the bacterial cells from 200 ml culture were washed twice in M9 media and sub-cultured into 1 L of M9 minimal medium at 37 °C for 5 h to de-repress expression of bio operon by starvation for biotin.  
    4. The bacteria were harvested by centrifugation (3,600 rpm, 16 min), washed three times with M9 medium, re-suspended in 1 ml of the same medium and mixed into 150 ml of the defined M9 agar media supplemented with 0.01% (w/v) TTC as a redox indicator.
    5. Finally, the mixture (5 ml per sector) was poured into Petri dishes sectored with plastic walls to avoid cross-feeding and a sterile paper disk (6 mm, BBL) was centered on the agar top of each sector.

  2. Preparation of cross-feeder strains
    1. In total, four feeder strains of A. tumefaciens corresponded to NTL4 (WT), FYJ283. (ΔbioBFDA), FYJ212 (ΔbioRat) and FYJ341 (ΔbioR::Km+bioRbme).
    2. The biotin auxotroph strain FYJ283 was cultivated in 5 ml of M9 medium supplemented  with 1 nM biotin, whereas the other three strains were cultivated in 5 ml of biotin-free M9 minimal media overnight.
    3. Overnight cultures were collected by centrifugation (3,000 rpm, 10 min), washed three times using the M9 liquid medium, and transferred into 100 ml of biotin-free M9 media for 6 more hours of growth at 30 °C to deplete trace amounts of intracellular biotin in the biotin auxotroph strain FYJ283.
    4. Following three rounds of washing with same media, bacteria were resuspended in M9 media and their optical densities at 600 nM (OD600) were adjusted to 1.5. 20 μl of A. tumefaciens culture (OD600 = 1.0) was spotted on the paper disc, and incubated overnight at 30 °C.
    5. Finally, the red deposit of formazan (Lin et al., 2010; del et al., 1979; Feng et al., 2014) suggests that the indicator strain ER90 is fed by the A. tumefaciens strains (seen in Figure 1), and the area size (square centimeters) of formazan represents the level of biotin pool produced by the different feeder strains.


    Figure 1. A representative photograph illustrating the relevance of BioR-mediated regulation to biotin synthesis. It was fully adapted from Feng et al. (2013a). Four A. tumefaciens strains cross-feed E. coli strain ER90 with deletion of full bio operon, which are NTL4 (WT), FYJ283 (ΔbioBFDA), FYJ212 (ΔbioRat), and FYJ 341 (ΔbioRat+bioRbme), respectively.

Recipes

  1. M9 minimal medium
    6 g of Na2PO4, 3 g of KH2PO4, 0.5 g of NaCl and 1 g of NH4Cl per liter

Acknowledgments

This protocol was adapted/modified from previous works seen in del Campillo-Campbell et al. (1079); Feng et al. (2014); Feng et al. (2013a); Feng et al. (2013b) and Lin et al. (2010).

References

  1. del Campillo-Campbell, A., Dykhuizen, D. and Cleary, P. P. (1979). Enzymic reduction of d-biotin d-sulfoxide to d-biotin. Methods Enzymol 62: 379-385.
  2. Feng, Y., Napier, B. A., Manandhar, M., Henke, S. K., Weiss, D. S. and Cronan, J. E. (2014). A Francisella virulence factor catalyses an essential reaction of biotin synthesis. Mol Microbiol 91(2): 300-314.
  3. Feng, Y., Xu, J., Zhang, H., Chen, Z. and Srinivas, S. (2013a). Brucella BioR regulator defines a complex regulatory mechanism for bacterial biotin metabolism. J Bacteriol 195(15): 3451-3467.
  4.  Feng, Y., Zhang, H. and Cronan, J. E. (2013b). Profligate biotin synthesis in α‐proteobacteria–a developing or degenerating regulatory system? Mol Microbiol 88(1): 77-92.
  5. Lin, S., Hanson, R. E. and Cronan, J. E. (2010). Biotin synthesis begins by hijacking the fatty acid synthetic pathway. Nat Chem Biol 6(9): 682-688.

简介

进行基于生物素生物测定的交叉进料实验,以阐明在根癌土壤杆菌(根瘤土壤杆菌)中通过生物素表达对生物素产生的影响。 (Feng et al。,2013)。本文使用的指示菌株是大肠杆菌(大肠杆菌)的生物素营养缺陷型菌株,ER90(ΔbioF bioC bioD ),其是由交叉进料。 tumefaciens 物种(Feng等人,2013a)。如其他人和我们的研究组所述配制不含生物素的M9基本培养基平板(Feng等人,2013b; Lin等人,2010; del > et al。,1979)。值得注意的是,将0.01%(w/v)氧化还原指示剂2,3,5-三苯基四唑氯化物(TTC)补充到上述介质中。因此,通过将TTC还原成不溶性红色甲was,观察到生物素的产生/产生,这是由于通过A进料的ER90生长。 tumefaciens 菌株(Feng等人,2014)。详细程序描述如下。

材料和试剂

  1. 四个。 根瘤土壤杆菌菌株[NTL4(WT),FYJ283(ΔbioBFDA),FYJ212(ΔbioR at)和FYJ341(ΔbioR :: em> Km + bioR bme)]
  2. E的生物素营养缺陷型菌株。 大肠杆菌,ER90
  3. 1nM生物素(Sigma-Aldrich,目录号:B4510)
  4. 0.01%(w/v)2,3,5-三苯基四唑氯化物(TTC)(AMRESCO,目录号:0765)
  5. 0.1%无维生素的酪蛋白氨基酸水解产物(Sigma-Aldrich,目录号:C7710)
  6. MgSO 4 4 /
  7. 葡萄糖
  8. M9基本培养基(见配方)

设备

  1. 离心机
  2. 培养皿(90mm)(Thermo Fisher Scientific,目录号:502VF)
  3. 无菌纸片(6mm,BBL)

程序

  1. 生物素生物测定板的制备
    1. 生物素测定板如前所述制备,几乎没有变化,其中之一涉及我们滴落纸碟的板琼脂的较薄厚度(Feng等人,2013b; Lin等人 al。,2010)。  
    2. 通过离心(3,600rpm,16分钟)收集菌株ER90的过夜培养物(在6ml具有1nM生物素的限定的M9基本培养基中生长,30℃),用相同体积(6ml)的M9培养基 并在含有1nM生物素的200ml M9基本培养基中在37℃至0.8OD 600培养。
    3. 为了除去过量的生物素,来自200ml培养物的所有细菌细胞在M9培养基中洗涤两次,并在37℃下在1L的M9基本培养基中继代培养5小时以解除抑制生物素的表达, em> operon by starvation for biotin。  
    4. 通过离心(3600rpm,16分钟)收获细菌,用M9培养基洗涤三次,重悬于1ml相同的培养基中,并混合到150ml限定的M9琼脂培养基中,补充有0.01%(w/v )TTC作为氧化还原指示剂。
    5. 最后,将混合物(每个部分5ml)倒入用塑料壁分成的培养皿中,以避免交叉进料,并将无菌纸盘(6mm,BBL)置于每个部分的琼脂顶部的中心。
  2. 交叉进料菌株的制备
    1. 总共,四个饲养株菌株。 tumefaciens 对应于NTL4(WT),FYJ283。 (Δ bioBFDA ),FYJ212(Δ bioR at)和FYJ341(Δ bioR :: Km + 。
    2. 将生物素营养缺陷型菌株FYJ283培养在5ml M9培养基中,与1nM生物素,而其他三个菌株在5ml无生物素的M9基本培养基中培养过夜。
    3. 通过离心(3,000rpm,10分钟)收集过夜培养物,使用M9液体培养基洗涤三次,并转移到100ml无生物素的M9培养基中,在30℃下再生长6小时以消耗痕量的细胞内生物素在生物素营养缺陷型菌株FYJ283中的生物素
    4. 在用相同培养基洗涤三轮后,将细菌重悬于M9培养基中,并将其在600nM(OD 600)的光密度调节至1.5。 20微升的A。将根癌土壤培养物(OD 600 = 1.0)点在纸碟上,并在30℃下孵育过夜。
    5. 最后,甲的红色沉积物(Lin等人,2010; del ,1979; Feng等人,2014)表明指示器应变ER90由ΔE馈送。 tumefaciens 菌株(见图1),而formazan的面积大小(平方厘米)表示由不同饲养菌株产生的生物素库水平。


    图1.代表性照片说明了BioR介导的调节与生物素合成的相关性。它完全改编自Feng等人(2013a)。 四个。 tumefaciens 菌株交叉进料。 (WT),FYJ283(Δ bioBFDA ),FYJ212(Δ bioR 操纵子缺失的大肠杆菌菌株ER90, em> at)和FYJ 341(Δ bioR 在+ bioR bme)。

食谱

  1. M9基本培养基
    6g Na 2 PO 4 PO 4,3g KH 2 PO 4 PO 4,0.5g NaCl和1g Na 2 PO 4, g的NH 4 Cl Cl

致谢

该协议是从以前在Campillo-Campbell等人(1079)中观察到的作品改编/修改的; Feng et al。(2014); Feng et al。 (2013a); Feng等人(2013b)和Lin等人(2010)。

参考文献

  1. del Campillo-Campbell,A.,Dykhuizen,D。和Cleary,P.P。(1979)。 将d-生物素d-亚砜酶促还原为d-生物素。 Methods Enzymol 62:379-385。
  2. Feng,Y.,Napier,B.A.,Manandhar,M.,Henke,S.K.,Weiss,D.S。和Cronan,J.E。 Francisella 毒力因子催化生物素合成的必要反应。 a> Mol Microbiol 91(2):300-314。
  3. Feng,Y.,Xu,J.,Zhang,H.,Chen,Z.and Srinivas,S。(2013a)。 Brucella BioR调节剂定义了细菌生物素代谢的复杂调节机制。/a> J Bacteriol 195(15):3451-3467
  4.   Feng,Y.,Zhang,H. and Cronan,J.E。(2013b)。 在α-变形菌细胞中利用生物素合成 - 发展中的或退化的调节系统? Mol Microbiol 88(1):77-92。
  5. Lin,S.,Hanson,R.E.and Cronan,J.E。(2010)。 生物素合成开始于劫持脂肪酸合成途径。 6(9):682-688。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Feng, Y., Xu, J., Zhang, H., Chen, Z. and Srinivas, S. (2014). Protocol for Biotin Bioassay-based Cross Feeding. Bio-protocol 4(18): e1242. DOI: 10.21769/BioProtoc.1242.
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