巴斯德毕赤酵母不同生长阶段内参基因的筛选与验证

【背景】巴斯德毕赤酵母(Komagataella phaffii)是一种甲基营养型酵母,近年来作为生产重组蛋白和构建生物合成途径的细胞工厂受到广泛关注。实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)是巴斯德毕赤酵母表达系统研究中一种快速、高效的基因表达水平检测技术,但需要进行归一化处理才能保证所得结果的可靠性。【目的】筛选并验证巴斯德毕赤酵母在不同生长阶段最稳定的内参基因用于精准归一化RT-qPCR的结果。【方法】通过转录组数据分析初步筛选出16个候选内参基因(rps8b、rpl35a、rpl10、eif5a、rpl19a、por1、rpl23b、0887、tif1、ole1、rpl14b、gs、sun、sdh2、trx1和ccp1)。通过RT-qPCR技术得到候选内参基因的C_t值,利用qBASE软件中的geNorm程序综合NormFinder算法评估内参基因的表达稳定性。【结果】通过geNorm分析得出精准归一化所需的最佳内参基因个数为2,最稳定的基因是rpl19a和tif1,NormFinder分析得到稳定性最高的内参基因为tif1。此外,利用甲酸脱氢酶编码基因fdh和乙醇脱氢酶甲醛脱氢酶双功能酶的编码基因afdh对候选内参基因进行验证。【结论】巴斯德毕赤酵母不同生长阶段的RT-qPCR进行精准归一化需要tif1和rpl19a这2个内参基因,为相关功能基因的表达定量提供了可靠的分析依据,补充了RT-qPCR分析中的内参基因,为巴斯德毕赤酵母不同生长阶段的基因表达调控及其应用研究提供了新的参考。     

小肠结肠炎耶尔森菌对多粘菌素B的压力应答

【背景】小肠结肠炎耶尔森菌(Yersinia enterocolitica)是重要的人畜共患食源性病原菌。由于其生存环境与传染性生活方式,小肠结肠炎耶尔森菌暴露在各种生存压力中,而胞膜压力应答能力对维持其环境耐受性和毒力发挥着重要作用。【目的】探究小肠结肠炎耶尔森菌在胞膜压力应答中的调节机制。【方法】通过使用多粘菌素B破坏小肠结肠炎耶尔森菌细胞膜的稳定性,并从生长能力、运动能力、生物被膜形成能力以及相关基因表达的变化探讨Rcs (Regulator of Capsule Synthesis)系统对多粘菌素B产生的胞膜压力的应答。【结果】多粘菌素B引起的细胞胞膜压力抑制了小肠结肠炎耶尔森菌的运动和生物被膜形成能力;而阻断Rcs信号途径后,小肠结肠炎耶尔森菌的运动和生物被膜形成能力有所恢复。对flhC、hmsS、hmsT等关键下游表型基因的表达水平的分析结果表明Rcs双组分系统对由多粘菌素B诱导的胞膜压力作出响应,通过感知胞膜胁迫向胞内传递信号,积极地调控细菌增强对抗菌肽的抗性。【结论】明确了Rcs双组分系统在响应多粘菌素B压力胁迫中的特异性调控作用,加深了对小肠结肠炎耶尔森菌环境应答机制的认识。     

Note on the fusion frequency for stimuli of alternating duration

The case of alternating stimulus patterns of moderately high intensity is considered in terms of the model previously discussed (Landahl, 1957,Bull. Math. Biophysics,19, 157–62). If both of the alternating patterns have the same light-dark ratio, then the relation between the period of the longer and the period of the shorter pattern at the critical flicker frequency is independent of the light-dark ratio and is given by a dimensionless expression which is roughly in agreement with data in which the light-dark ratio is one (C. R. Brown and D. M. Forsyth, 1959,Science,129, 390–91). This research was supported in whole or in part by the U. S. Air Force under Contract AF 49(638)-414 monitored by the Air Force Office of Scientific Research.     

Some remarks on the central nervous system

It is pointed out that the successes obtained in the mathematical biology of the central nervous system are based mostly on a number of more or less complicated neuronic circuit models, each inventedad hoc for the purpose of explaining a given phenomenon. The individual models remain disconnected from each other, however, and the unity of the CNS is not apparent. (Rashevsky,Mathematical Biophysics, 3rd Edition, Vol. II, 1960. New York, Dover Publications, Inc.) Some “field theories” of the CNS, as for example that of Griffith (Bull. Math. Biophysics,25, 111–120, 1963;27, 187–195, 1965), give more expression to this unity but lose in the explanation of specific phenomena. The present paper starts with the picture thatevery neuron in the brain isdirectly or indirectly affected to some extent byevery other neuron. This leads to a system of equations with a very large number of variables. Such a system can be replaced in the limiting case by an integral equation of the first kind. At least two specific results can be obtained with this approach and suggestions for further improvement are made.     

挖掘与调控乙酸胁迫响应基因提高重组酿酒酵母合成番茄红素水平

【背景】乙酰辅酶A是酿酒酵母异源合成番茄红素的重要中间产物,胞质中乙酰辅酶A主要来自乙酰辅酶A合成酶催化乙酸合成。【目的】通过外源添加乙酸盐结合调控乙酸胁迫应答基因增加胞内乙酰辅酶A含量,改善细胞生长,促进番茄红素合成。【方法】在合成番茄红素的重组酵母菌中过表达乙酰辅酶A合成酶编码基因(acs2),在发酵过程中添加10g/L乙酸盐,结合转录组学分析挖掘乙酸胁迫响应基因,进行单一和组合调控。【结果】添加乙酸盐后,重组菌Y02中番茄红素含量增加了19.14%,但细胞生长受到抑制,转录组学结果表明adk2、fap7、hem13、elo3、pdc5、set5、pmt5、hst4、clb2和swe1表达水平增加,因此构建了单基因和双基因过表达菌株,其中Y02-set5-hst4菌在添加乙酸盐后细胞生长得到了显著改善,同时胞内乙酰辅酶A浓度提高了78.21%,番茄红素含量和产量达到12.62 mg/g-DCW和108.67 mg/L,与对照菌Y02相比分别提高了42.76%和67.13%。同时该菌中甲羟戊酸途径中关键基因erg12、erg20和hmg1的表达量与对照菌相比分别上调了1.70、1.4...     

改造大肠杆菌卟啉代谢途径对重组过氧化物酶活性的影响

【目的】原核表达某些需辅因子的外源蛋白时往往酶活偏低,为提高酶活和减少外加辅因子的成本,我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。【方法】本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD(catalase-peroxidase)编码基因kat G的ORF,构建原核表达载体p ET28a-kat G,实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素,而血卟啉是血红素的骨架,通过构建原核表达载体p UC19-tac-hem A,将编码5-氨基乙酰丙酸合成酶的hem A基因在大肠杆菌中过量表达,提高卟啉的含量,从而提高重组蛋白CAT-POD的酶活。【结果】最终的CAT酶活达到了377 U/m L,为对照组的7.5倍。【结论】本研究为工业生产高活性CAT-POD提供了有效的方案,也为体外重组表达含辅因子的蛋白提供可借鉴的思路。     

解淀粉芽孢杆菌内源启动子的筛选及表达碱性果胶酶的应用研究

【目的】通过检测目的基因的转录水平和表达强度,筛选来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的内源启动子,确定适合碱性果胶酶基因表达的强启动子,并进一步对选用的强启动子进行分析。【方法】通过生物信息学手段对启动子片段进行预测与筛选,采用相对荧光强度、酶活力等表征手段进行分析,同时采用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, RT-qPCR)技术检测不同启动子的转录水平。【结果】启动子PrapA、PmetE-1、Phin-1表达碱性果胶酶的活力分别是P43启动子的9.8倍、4.8倍、3.0倍,筛选出的这3个强启动子为其他异源基因在解淀粉芽孢杆菌中的表达奠定了基础。【结论】通过生物信息学手段预测及筛选启动子,筛选得到比较强的启动子PrapA、PmetE-1和Phin-1,有效提高了碱性果胶酶的表达量。     

Recent Advances in GFP Folding Reporter and Split-GFP Solubility Reporter Technologies. Application to Improving the Folding and Solubility of Recalcitrant Proteins from Mycobacterium tuberculosis

We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691–695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102–107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.     

IL7R gene expression network associates with human healthy ageing

Background

The level of expression of the interleukin 7 receptor (IL7R) gene in blood has recently been found to be associated with familial longevity and healthy ageing. IL7R is crucial for T cell development and important for immune competence. To further investigate the IL7R pathway in ageing, we identified the closest interacting genes to construct an IL7R gene network that consisted of IL7R and six interacting genes: IL2RG, IL7, TSLP, CRLF2, JAK1 and JAK3. This network was explored for association with chronological age, familial longevity and immune-related diseases (type 2 diabetes, chronic obstructive pulmonary disease and rheumatoid arthritis) in 87 nonagenarians, 337 of their middle-aged offspring and 321 middle-aged controls from the Leiden Longevity Study (LLS).

Results

We observed that expression levels within the IL7R gene network were significantly different between the nonagenarians and middle-aged controls (P?=?4.6 × 10^?4), being driven by significantly lower levels of expression in the elderly of IL7, IL2RG and IL7R. After adjustment for multiple testing and white blood cell composition and in comparison with similarly aged controls, middle-aged offspring of nonagenarian siblings exhibit a lower expression level of IL7R only (P?=?0.006). Higher IL7R gene expression in the combined group of middle-aged offspring and controls is associated with a higher prevalence of immune-related disease (P?=?0.001). On the one hand, our results indicate that lower IL7R expression levels, as exhibited by the members of long-lived families that can be considered as ‘healthy agers’, are beneficial in middle age. This is augmented by the observation that higher IL7R gene expression associates with immune-related disease. On the other hand, IL7R gene expression in blood is lower in older individuals, indicating that low IL7R gene expression might associate with reduced health. Interestingly, this contradictory result is supported by the observation that a higher IL7R gene expression level is associated with better prospective survival, both in the nonagenarians (Hazard ratio (HR)?=?0.63, P?=?0.037) and the middle-aged individuals (HR?=?0.33, P?=?1.9 × 10^–4).

Conclusions

Overall, we conclude that the IL7R network reflected by gene expression levels in blood may be involved in the rate of ageing and health status of elderly individuals.

     

Probability distributions associated with distinct hits on targets

Some probability distributions connected with distinct hits on targets, using two different firing schemes, are developed. It is assumed that any shot has a probabilityp, not necessarily unity, of hitting the target at which it was aimed. The development uses a well-known expression for the probability that exactlyt ofN possible events occur simultaneously. Some of the formulae developed here include as special cases the probabilities derived separately and by more complicated arguments in papers by N. Rashevsky. (Bull. Math. Biophysics,17, 45–50, 1955) and A. Rapoport (Bull. Math. Biophysics,13, 133–38, 1951).     

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

Objectives

Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid.

Results

The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD.

Conclusions

These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

     

An evolution of pulse speed in arteries

In this work, treating the artery as a thick-walled cylindrical shell made of an incompressible, isotropic and elastic solid, utilizing the large deformation theory and the stress-strain relation proposed by Demiray (1976b,Trans. ASME Ser. E, J. Appl. Mech.,98, 194–197), an explicit expression for the pulse speed is obtained and the effect of lumen pressure and the axial stretch on wave speed is discussed. Numerical results indicate that the wave speed increases with lumen pressure but decreases with the axial stretch. The results of the present model are compared with our previous work (Demiray, 1988,J. Biomech. 21, 55–58) on the same subject.     

Linear bulk diffusion into heterogeneous tissue

In this paper an expression is derived which describes the transient overall uptake of an inert solute by a section of tissue excised with parallel faces and placed upon an impermeable base. The approach diverges from the conventional analyses for perfused tissue (Morales and Smith,Bull. Math. Biophysics,6, 125–141, 1944;7, 47–99, 1945) because the extravascular zone is regarded as a heterogeneous diffusion medium. Account for this is taken by regarding tissue as effectively composed of two phases—a continuous (extracellular) phase similar to water, and a dispersed phase comprising cells of irregular profile. In both phases the relevant mode of uptake is taken as bulk diffusion rather than surface permeation, thus emphasizing the influence of the internal geometry of the tissue upon its overall exchange response.     

熊蜂生假丝酵母启动子的筛选及强度分析

【背景】熊蜂生假丝酵母(Starmerella bombicola)作为一种非常规假丝酵母菌株,因其具备高产槐糖脂生物表面活性剂的能力而受到广泛关注。然而,由于自身的表达系统并不完善,限制了该菌株的代谢工程改造。【目的】克隆、筛选及鉴定新的系列内源启动子表达元件。【方法】通过对比分析熊蜂生假丝酵母全基因组及9种功能已知目的基因信息,并结合启动子预测网站,筛选获得系列启动子候选序列,以SbGFP (密码子优化后的酵母增强型绿色荧光蛋白)为报告基因进行整合表达,通过绿色荧光蛋白强度及转录水平分析鉴定启动子强度。【结果】在分别以葡萄糖和油酸作为唯一碳源的条件下,启动子PTEF1和PGPD在不同碳源培养条件下均显示出较高的转录水平。启动子PCYP52M1、PUGTA1、PUGTB1及PMOB在以油酸为唯一碳源时具有弱转录活性,而在以葡萄糖为唯一碳源时则未检测到它们具有转录活性,推测它们是油酸诱导型启动子。进一步利用实时荧光定量PCR (RT-qPCR)对SbGFP进行转录水平分析,检测结果与绿色荧光表达水平一致。【结论】获得了系列熊蜂生假丝酵母内源性启动子,进一步丰富了该菌株的表达元件,为菌株的代谢工程改造及基因的表达与调控奠定了理论基础。     

利用CRISPRi挖掘大肠杆菌生物合成D-泛酸的关键基因

【目的】D-泛酸(D-pantothenic acid,DPA)是一种重要的功能化合物,被广泛应用于医疗保健、化妆品、动物食品和饲料等领域,具有良好的市场前景及应用。本研究以实验室保藏的大肠杆菌菌株DPAP10为底盘菌株,利用CRISPR干扰(clustered regularly interspaced palindromic repeats interference,CRISPRi)技术,筛选影响工程菌株DPA生物合成的内源性基因靶点。【方法】构建了p Target和pd Cas9的双质粒CRISPRi系统,可以实现对基因单个或组合表达抑制,摇瓶发酵检测基因抑制对DPA合成的影响;通过实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-q PCR)检测了基因抑制后的转录水平;通过高效液相色谱(high performance liquid chromatography,HPLC)检测了中间代谢物分析代谢通路变化。【结果】成功从126个靶基因中筛选得到5个显著影响DPA合成的关键...     

假结核耶尔森氏菌中Phd-Doc毒素-抗毒素系统的功能鉴定

【背景】毒素-抗毒素系统在微生物体内广泛存在,在微生物对抗外界不良环境方面发挥重要作用。【目的】以模式细菌假结核耶尔森氏菌(Yersinia pseudotuberculosis,Yptb)为材料,探究其编码的Phd-Doc毒素-抗毒素系统的作用机制和生物学功能。【方法】通过生物信息学方法预测Yptb中编码的Phd-Doc毒素-抗毒素系统,通过毒性分析、基因表达分析及蛋白相互作用对其进行鉴定;通过抗生素胁迫、氧胁迫、生物被膜形成等实验研究Phd-Doc在体内发挥的生物学功能。【结果】生物信息学分析鉴定出一对Phd-Doc毒素-抗毒素系统,发现二者共转录且相互作用;毒素蛋白Doc能够引起大肠杆菌形态发生变化并抑制其生长,抗毒素蛋白Phd能中和Doc的毒性;Phd-Doc毒素-抗毒素系统具有自调控抑制效应;phd-doc的缺失对Yptb自身的生长无影响,而且毒素蛋白Doc在野生型Yptb内过表达并未显示毒性;phd-doc在转录水平上响应了抗生素胁迫和氧胁迫,其中,对氯霉素胁迫最为敏感,但并不影响Yptb的生长;同时,Phd-Doc能够影响Yptb的生物被膜形成能力。【结论】Yptb中Phd-Doc毒素-抗毒素系统的功能鉴定对于更好地了解在多变的外部环境下微生物的定殖和响应机制具有重要意义。