Discovery of ligands for beta gamma subunits from phage-displayed peptide libraries.
Discovery of ligands for beta gamma subunits from phage-displayed peptide libraries.
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从噬菌体展示肽库中发现 β γ 亚基的配体。
DOI：
10.1016/s0076-6879(02)44740-4
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发表时间：
2002
影响因子：
--
通讯作者：
Scott,JamieK
中科院分区：
文献类型：
--
作者：
Smrcka,AlanV;Scott,JamieK
方法制备饥饿K91大肠杆菌。用f88-4-丝状噬菌体感染大肠杆菌细胞，显示由F附加体编码的性菌毛的大肠杆菌菌株。下面的程序最大限度地展示F菌毛，并浓缩细胞以获得最佳感染。大肠杆菌细胞（CGSC#4616; http：//cgsc.org.cn/）。生物学耶鲁edu）从甘油储备液中分离到NZY琼脂上，并在37 ℃孵育过夜。从平板上挑取一个分离的菌落，并转移到含有2 ml NZY培养基的14 ml卡扣盖培养管中。将培养物在振荡器中以250 rpm在37 ℃孵育过夜。早晨，在125 ml烧瓶中加入20 ml NZY培养基和200 µl过夜培养物。在37 ℃下以250 rpm振摇，直至细胞达到0.45的OD 600（约1小时20分钟后开始测量OD）。将振荡器速度降至100 rpm，持续10-20分钟。再次测量OD 600，其应在0.5至0.6之间。将培养物转移至50 ml无菌锥形管中，并在4 ℃或室温下以600 g离心10 min。将细胞悬浮于20 ml无菌80 mM NaCl中，并在125 ml培养瓶中于37 ℃下以100 rpm振摇45 min，使细胞饥饿。将混合物转移至50 ml无菌锥形管中，并在4 ℃下以850 g离心10 min。将细胞轻轻悬浮于1 ml 4 NAP缓冲液中。将细胞保存在冰上直至准备使用。细胞可以在冰上储存长达1周，但最好在新鲜制备时使用。
MethodsPreparation of Starved K91 E. coli Cells for Infection with f88-4—Filamentous phage infect E. coli strains displaying the sex pilus encoded by the F episome. The following procedure maximizes display of the F pilus and concentrates the cells for optimal infection.Streak K91 strain of E. coli cells (CGSC# 4616; http://cgsc. biology. yale. edu) from a glycerol stock onto NZY agar and incubate overnight at 37. Pick an isolated colony from the plate and transfer to a 14 ml snap-cap culture tube containing 2 ml of NZY medium. Incubate the culture overnight at 37 in a shaker at 250 rpm. In the morning inoculate 20 ml of NZY medium with 200 µl of the overnight culture in a 125 ml flask. Shake at 250 rpm at 37 until the cells reach OD600 of 0.45 (start to measure the OD after about 1 h and 20 min). Reduce the speed of the shaker to 100 rpm for 10–20 min. Measure the OD600 again, which should be between 0.5 and 0.6. Transfer the culture to a 50 ml sterile conical tube and centrifuge at 600g for 10 min at 4 or room temperature. Suspend the cells in 20 ml of sterile 80 mM NaCl and shake at 100 rpm in a 125 ml culture flask at 37 for 45 min to starve the cells. Transfer the mixture to a 50 ml sterile conical tube and centrifuge at 850g for 10 min at 4. Gently suspend the cells in 1 ml of 4 NAP buffer. Keep the cells on ice until ready for use. The cells can be stored on ice for up to 1 week but are best if used when freshly prepared.