In situ structure of the complete Treponema primitia flagellar motor

The bacterial flagellar motor is an amazing nanomachine: built from approximately 25 different proteins, it uses an electrochemical ion gradient to drive rotation at speeds of up to 300 Hz (refs 1, 2). The flagellar motor consists of a fixed, membrane-embedded, torque-generating stator and a typically bidirectional, spinning rotor that changes direction in response to chemotactic signals. Most structural analyses so far have targeted the purified rotor, and hence little is known about the stator and its interactions. Here we show, using electron cryotomography of whole cells, the in situ structure of the complete flagellar motor from the spirochaete Treponema primitia at 7 nm resolution. Twenty individual motor particles were computationally extracted from the reconstructions, aligned and then averaged. The stator assembly, revealed for the first time, possessed 16-fold symmetry and was connected directly to the rotor, C ring and a novel P-ring-like structure. The unusually large size of the motor suggested mechanisms for increasing torque and supported models wherein critical interactions occur atop the C ring, where our data suggest that both the carboxy-terminal and middle domains of FliG are found.     

大肠杆菌JM109菌株高效电转化条件的研究

通过感受态细胞的生长状态、转化温度及不同的电击杯内径、电压等影响电转化的重要条件,探讨索了大肠杆菌JM109菌株电转化的最优条件.在感受态细胞OD600值为0.5-0.7,转化环境温度为4℃,电容25 μF、并联电阻200 Ω、电压2.5 kV和0.2 cm电击杯电转化效率最高,可达到9.12×108.     

Direct observation of steps in rotation of the bacterial flagellar motor

The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel many species of swimming bacteria. The rotor is a set of rings up to 45 nm in diameter in the cytoplasmic membrane; the stator contains about ten torque-generating units anchored to the cell wall at the perimeter of the rotor. The free-energy source for the motor is an inward-directed electrochemical gradient of ions across the cytoplasmic membrane, the protonmotive force or sodium-motive force for H+-driven and Na+-driven motors, respectively. Here we demonstrate a stepping motion of a Na+-driven chimaeric flagellar motor in Escherichia coli at low sodium-motive force and with controlled expression of a small number of torque-generating units. We observe 26 steps per revolution, which is consistent with the periodicity of the ring of FliG protein, the proposed site of torque generation on the rotor. Backwards steps despite the absence of the flagellar switching protein CheY indicate a small change in free energy per step, similar to that of a single ion transit.     

Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.     

基于FPGA的等占空比的整数分频器设计

在复杂逻辑电路设计中,经常会需要多个不同频率的信号,因而系统本身的震荡源就不能满足设计的要求.本文给出了一种可以实现等占空比任意整数的分频方法,并以8分频和9分频为例,介绍了在QUARTUS软件下,利用VHDL硬件描述语言来设计分频器的方法.程序通过仿真和测试,实验结果符合设计要求.     

Firing patterns of motor units in normal rats

Skeletal muscles consist of motor units which may differ considerably in contractile properties and types of usage. Some units participate mainly in relatively rare, quick movements and contract rapidly and are easily fatigued (type FF); others contribute to the maintenance of posture and hence contract slowly and are fatigue-resistant (type S), while others are both fast and fatigue-resistant (type FR). Our understanding of motor control mechanisms and the dependence of contractile properties on usage would be enhanced if more quantitative information were available concerning the firing patterns of individual motor units during normal motor behaviour. Therefore, we have made continuous recordings for extended periods from single motor units in the fast extensor digitorum longus (edl) and the slow soleus (sol) muscle of freely moving adult rats. By counting the total number of discharges for each unit, and by determining the distributions of interspike intervals and the duration of the individual impulse trains, we have obtained information about firing rate, amount of use, modulation of muscle force and tonic and phasic behaviour for 16 motor units. We now report that these units fall into three classes apparently corresponding to type FF and FR in the edl muscle and type S in the soleus muscle.     

类人胶原蛋白基因工程茵高密度发酵过程控制

目的优化基因工程菌Escherichia coli BL21生产人源型胶原蛋白的高密度发酵工艺。方法建立一套以溶解氧浓度和比生长速率为中心的控制策略，采用溶氧反馈或pH反馈调节补料速度。结果OD600可达150，胶原蛋白的表达量为9g／L。结论20％～30％饱和度的溶解氧和0．1-0．2h^-1的比生长速率有利于细胞生长和产物表达，其与降低乙酸等有害代谢副产物的积累密切相关。     

类人胶原蛋白基因工程菌高密度发酵过程控制

目的优化基因工程菌Escherichia coli BL 21生产人源型胶原蛋白的高密度发酵工艺。方法建立一套以溶解氧浓度和比生长速率为中心的控制策略,采用溶氧反馈或pH反馈调节补料速度。结果 OD600可达150,胶原蛋白的表达量为9g／L。结论 20％-30％饱和度的溶解氧和0．1- 0．2 h-1的比生长速率有利于细胞生长和产物表达,其与降低乙酸等有害代谢副产物的积累密切相关。     

Construction of a genetic toggle switch in Escherichia coli

It has been proposed' that gene-regulatory circuits with virtually any desired property can be constructed from networks of simple regulatory elements. These properties, which include multistability and oscillations, have been found in specialized gene circuits such as the bacteriophage lambda switch and the Cyanobacteria circadian oscillator. However, these behaviours have not been demonstrated in networks of non-specialized regulatory components. Here we present the construction of a genetic toggle switch-a synthetic, bistable gene-regulatory network-in Escherichia coli and provide a simple theory that predicts the conditions necessary for bistability. The toggle is constructed from any two repressible promoters arranged in a mutually inhibitory network. It is flipped between stable states using transient chemical or thermal induction and exhibits a nearly ideal switching threshold. As a practical device, the toggle switch forms a synthetic, addressable cellular memory unit and has implications for biotechnology, biocomputing and gene therapy.     

大肠杆菌NADP特异性谷氨酸脱氢酶基因的克隆及其在大肠杆菌中的高效表达与纯化

谷氨酸脱氢酶是生物体内最主要的氧化脱氨基酶类，为了进一步研究其功能，我们以大肠杆菌DH5α菌株基因组DNA为模板和相应的寡聚脱氧核苷酸为引物，进行PCR扩增大肠杆菌NADP特异性谷氨酸脱氢酶基因(NADP-specific glutamate dehydrogenase，gdhA gene)，将所得DNA片断连接到质粒pUC18上，转化大肠杆菌DH5α，进行蓝白筛选和酶切鉴定，经测序证明序列正确无误后将gdhA基因连接到表达载体pTrcHisC上，在大肠杆菌BL21(DE3)中表达，经SDS-PAGE和双波长扫描分析，确定大肠杆菌谷氨酸脱氢酶在大肠杆菌中表达时以包涵体的形式存在，未能检测到可溶性蛋白的表达，表达量可达菌体总蛋白的15％以上．     

产甘油重组大肠杆菌的构建及其耐高渗透压性能的测定

克隆了酿酒酵母(Saccharomyces cerevisiae) 中的3-磷酸甘油脱氢酶和3-磷酸甘油酯酶基因,通过融合PCR构建了双基因共表达载体,将酵母细胞内应答渗透压变化的甘油合成途径引入大肠杆菌（Escherichia coli）。以葡萄糖为底物对重组大肠杆菌进行摇瓶发酵培养,该重组菌的甘油产量为1g/L。渗透压胁迫测试证明该重组菌的耐渗透压性能较出发菌株有明显提高。     

Escherichia coli swim on the right-hand side

The motion of peritrichously flagellated bacteria close to surfaces is relevant to understanding the early stages of biofilm formation and of pathogenic infection. This motion differs from the random-walk trajectories of cells in free solution. Individual Escherichia coli cells swim in clockwise, circular trajectories near planar glass surfaces. On a semi-solid agar substrate, cells differentiate into an elongated, hyperflagellated phenotype and migrate cooperatively over the surface, a phenomenon called swarming. We have developed a technique for observing isolated E. coli swarmer cells moving on an agar substrate and confined in shallow, oxidized poly(dimethylsiloxane) (PDMS) microchannels. Here we show that cells in these microchannels preferentially 'drive on the right', swimming preferentially along the right wall of the microchannel (viewed from behind the moving cell, with the agar on the bottom). We propose that when cells are confined between two interfaces--one an agar gel and the second PDMS--they swim closer to the agar surface than to the PDMS surface (and for much longer periods of time), leading to the preferential movement on the right of the microchannel. Thus, the choice of materials guides the motion of cells in microchannels.     

Escherichia coli dam—dcm—菌株的研究进展

E.coli dam-dcm是一种在分子生物学技术中被广泛应用的菌株之一。Dam和Dcm是两种甲基转移酶，Dam识别GATC位点而Dcm识别CCA（T）GG位点[1]，在E.coli细胞的生命活动中，dam基因产物在DNA的错配修复中具有重要作用，另外，dam的甲基化作用和DNA的复制和基因表达的调节等细胞生命活动过程，Dcm甲基化的生物学功能在很短的补丁修复有很重要的作用。Streptomyces(链霉菌），Bacillus(芽胞杆菌）和Paracoccus（副球菌）的转化通过用dam和dcm位点没有甲基化的DNA可以得到很大的改善[6]。因为5－甲基胞嘧啶对肼有抗生，所以 从dcm缺陷的菌株分离到的DNA用Mazam和Gilbert法进行测序，结果更好[10]。