请提供整合克隆玛格丽特厘米史密斯 资金支持: 加拿大基因组/基因组大草原 NSERC发现和战略项目赠款

1。文化生产<ol><li>准备无菌液体介质:TY 10（5克/ L，蛋白胨，酵母提取物3克/升，0.44克/升的氯化钙脱水），11磅（10克/ L，蛋白胨，5 g / L酵母提取物，5克/氯化钠，pH值7）。 </li><li>从一个单一的殖民地:TY传媒SmUW227（ 根瘤菌的 LP应变:建设将在别处描述，紧张施工细节要求）（）到5毫升50微克/毫升大观接种。接种以下株，到5毫升的LB液体培养基，给予抗生素补充: 大肠杆菌大肠杆菌 DH5α12 pJC2，整合表达载体，含10微克/毫升四环素; E。大肠杆菌 MT616 13日的动员，10微克/毫升氯霉素; 和 E。 大肠杆菌 DH5αpJH110，捐助卡带载体，含有5微克/毫升的庆大霉素。 </li><li>孵育E。在37及隔夜大肠杆菌菌株度;ç不断晃动。孵育S。两天用颤抖的根瘤菌株在30°C。它有可能获得与根瘤菌文化具有较大的接种（1:500饱和文化的亚文化）过夜。 </li></ol> 2。文化的制备和混合<ol><li>每一株洗净文化1.5毫升离心收集细胞沉淀在17000 XG为30秒和1毫升无菌0.85％氯化钠重新悬浮;重复一次。 </li><li> 100无菌0.85％氯化钠液重悬沉淀。 </li><li>个别发现每一个普通的TY琼脂平板上的应变10微升水洗文化。这些点是控制点。 </li><li>加入40μL的每个菌株的大肠杆菌除外大肠杆菌 DH5α含有pJC2在无菌试管，混合，并当场这种混合物120μL纯TY琼脂平板上12。这点是没有整合阴性对照。 </li><li>加入40μl每一株的无菌ţUBE，千姿百态，与现货160μL纯TY琼脂平板上这种混合物。这点是IMCE交配点。 </li><li>允许点干层流罩。 </li><li>密封板和30°C过夜孵育。 </li></ol> 3。隔离跨稳定整合<ol><li>连续控制点和IMCE点到TY平板补充200微克/毫升链霉素（SmUW227菌株Rm1021基于它带有一个突变赋予高级别抗链霉素14）和30微克/毫升的庆大霉素。 </li><li>计算的IMCE效率的，悬浮约¼的的的IMCE交配点，在500μL无菌0.85％氯化钠。 10倍系列稀释至10-7。板稀释到10 10 -2 TY琼脂板补充200微克/毫升链霉素和30微克/毫升庆大霉素，选择跨稳定整合-5。板稀释度10 -3 THRough 10 -7补充200微克/毫升链霉素，选择反式稳定整合和潜在的受助人，即总受助TY琼脂板。 </li><li>对于悬浮隔离的无标记转稳定整合约500μL无菌0.85％氯化钠一季度IMCE交配点。 10倍系列稀释至10-6。板稀释补充200微克/毫升链霉素和200微克/毫升的X GLUC四个TY琼脂板10-4到10-6。 </li><li> 3天在30°C孵育板。 </li><li>查看RFP的反稳定整合在绿光（525纳米），并通过一个红色的过滤器（&gt; 610 nm）的15。 </li><li>计算％IMCE效率，反式稳定整合菌落形成单位相比，总受助人的菌落形成单位。大约一半的反式稳定整合将发生真正的磁带交换，使他们的白色和大观霉素敏感。 </li><li>找到刘元反稳定整合（W，捐助向量在这里不包含像pJH110）屏幕为白色菌落（延长潜伏期，在室温下的额外1-2天，将加强分化的殖民地通过X GLUC的的通用汽车研究，这是必要的， 在 S 根瘤菌 SmUW227），TY琼脂平板上确认没有殖民地的筛选抗生素和TY琼脂板含有100微克/毫升大观霉素的敏感性。 </li></ol> 4。代表结果经过3天的潜伏期上TY辅以链霉素和庆大霉素控制条纹应该没有增长。 IMCE连胜应该有头连胜和图2可以看出，第二连胜的许多殖民地的融合发展。跨国整合的效率，跨稳定整合总受助人的百分比表示，应该在0.5％的范围内。约一半跨稳定整合，将大观霉素敏感和白色显示他们已​​经经历了真正的磁带交换。绿光（525纳米）下，通过一个红色的过滤器（&gt; 610 nm）的15时，，横贯稳定整合包含从pJH110的测试RFP捐助卡带显示有迹可寻RFP的荧光。 <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="图1"/> 图1共轭混合插图:质粒计算机辅助从pRK600转移基因的表达，都是随机从细胞到细胞转移。在混合物中的反式稳定整合创造IMCE，整合（int）的助手的质粒pJC2和捐助质粒pJH110所需的两个质粒通过的LP应变的收购，这种转移的结果。从非复制捐助质粒（pJH110）捐助卡带交换通过ΦC31整合活动的LP，磁带在染色体上的标记，造成损失的LP标记（SPr和uidA基因 ）和捐助卡带（RFP和通用汽车研究）中产生的反式完整之维护。 <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="图2"/> 图2。答 :非选择性的TY板琼脂表面上呈现出干细胞的混合物。接合点板A:链霉素，庆大霉素-X GLUC的板从顶部挑染的接合点，左顺时针没有整合控制，将与 IMCE 根瘤菌，E.大肠杆菌 DH5α， 大肠杆菌含有pJC2控制大肠杆菌 DH5a含有pJH110控制，E.大肠杆菌 MT616控制，S.根瘤菌 UW227 控制 C:10 -2稀释交配点悬浮上TY链霉素-X GLUC琼脂ð:10 -2上:TY链霉素，庆大霉素- X-GLUC琼脂（蓝色菌落是单一的重组稀释交配点悬浮，白色菌落的真实经历磁带交换） 电子邮件 :10 -2的交配点悬浮TY链霉素-X GLUC的显示荧光不足的琼脂稀释传真:10 -2交配点悬浮TY链霉素，庆大霉素- X-GLUC两级琼脂稀释荧光（光明殖民地对应蓝色菌落，并具有较高的RFP表达大概是由于虽然读从向量序列，其中殖民地经历真正 ​​的盒式交换包含只有立即启动与招标书没有读通过lac启动子载体，这是不存在的。）。

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没有利益冲突的声明。

该的IMCE技术允许为一个单一的attB两侧LP轨迹以前工程菌的DNA磁带的有效整合。一旦RFP创建捐助盒克隆所需的构造，该技术并不需要随后的DNA纯化和改造，使得它非常健壮。这是至关重要的，包括适当的生长控制，是某些抗生素耐药性是由于建立反稳定整合，而不是其他因素。 IMCE生产效率约0.5％的反式稳定整合。相反，通过同源重组的双重交叉发生在约10 -6?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>链霉素</td><td> bioshop加拿大公司</td><td> STP101 </td><td></td></tr><tr><td>大观</td><td> bioshop加拿大公司</td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td>庆大霉素</td><td> bioshop加拿大公司</td><td> GTA202 </td><td></td></tr><tr><td>氯霉素</td><td> bioshop加拿大公司</td><td> CLR201 </td><td></td></tr><tr><td>四环素</td><td> bioshop加拿大公司</td><td> TET701 </td><td></td></tr><tr><td>卡那霉素</td><td> bioshop加拿大公司</td><td> KAN201 </td><td></td></tr><tr><td>细菌级琼脂</td><td&gt; Bioshop加拿大公司</td><td> AGR001 </td><td></td></tr><tr><td>蛋白胨</td><td> bioshop加拿大公司</td><td> TRP402 </td><td></td></tr><tr><td>酵母提取物</td><td> bioshop加拿大公司</td><td> YEX401 </td><td></td></tr><tr><td>氯化钠</td><td> bioshop加拿大公司</td><td> SOD001 </td><td></td></tr><tr><td>氯化钙</td><td> bioshop加拿大公司</td><td> CCL444 </td><td></td></tr><tr><td> X - GLUC </td><td>黄金生物技术公司</td><td> G1281C1 </td><td></td></tr><tr><td> 大肠杆菌 MT616应变</td><td>可要求</td><td></td><td>也可用于我们的实验室之外</td></tr><tr><td> 大肠杆菌 pJC2应变</td><td>在内部，可以通过请求</td><td></td><td></td></tr><tr><td> 大肠杆菌 pJH110应变</td><td>在内部，可以通过requesţ </td><td></td><td></td></tr><tr><td> SmUW227应变</td><td>在内部，可以通过请求</td><td></td><td></td></tr></tbody></table>

site-specific bacterial chromosome engineering: &#934;c31 integrase mediated cassette exchange (imce)

可用于细菌染色体的稳定保持在大型基地范围内1外源DNA。整合到染色体复制质粒，质粒稳定性，质粒不相容性和质粒拷贝数变异，如规避的问题。这种方法使用从链霉菌噬菌体（Φ）C31的2,3特定地点的整合。 ΦC31整合酶催化两个特定的DNA位点之间的直接重组:attB和attP（34和39个基点，分别）4。此重组是稳定的，并没有恢复5。一个"着陆垫"（LP）的1霉素抗性基因，AADA（SPR），和大肠杆菌组成的序列大肠杆菌 β-葡萄糖醛酸酶基因（uidA基因 ）两侧attP网站已整合到苜蓿根瘤菌，Ochrobactrum anthropi，在区域间的农杆菌染色体， 上午PC轨迹和的TETA轨迹，分别与根瘤菌用于在该协议。捐助动员的载体侧翼1填塞红色荧光蛋白（RFP）基因和抗生素抗性基因的attB网站也已建成。在这个例子中使用庆大霉素的耐药质粒pJH110。使用SPH我和 Pst一所需的结构可能会被替换的RFP基因6另外attB网站两侧，可能是一种人工合成的构造子克隆到动员的载体，如pK19mob 7。由lac启动子驱动的ΦC31整合（克隆pHS62 8）基因的表达质粒pRK7813 9上动员的广泛的寄主范围。 一个tetraparental交配协议用于转移到捐助盒的LP菌株，从而取代捐助卡带在LP序列标记。这些细胞的移植S-稳定整合。横贯稳定整合，形成了0.5％的典型效率。横贯稳定整合通常是发现在第一500-1000菌落筛选抗生素的敏感性，或用5 - 溴-4 - 氯-3 - 吲哚基-β-D-葡萄糖醛酸（X GLUC）蓝白筛选。该协议包含用于创建和隔离跨稳定整合的交配和选拔程序。

Watch this Scientific Journal Video about Site-specific Bacterial Chromosome Engineering: Î¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

描述一个快速和有效的方法融入预制受体菌株，称为停机坪株，外源DNA的利益。该方法允许进入工程着陆垫一个给定的应变轨迹的站点特定的DNA盒的整合，通过共轭ΦC31整合表达。

网站特定的细菌染色体工程:ΦC​​31整合介导的盒式交换（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

site-specific-bacterial-chromosome-engineering-c31-integrase-mediated

Research

JoVE Journal

Bioengineering

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

15.5K 次观看.  University of Waterloo. 描述一个快速和有效的方法融入预制受体菌株，称为停机坪株，外源DNA的利益。该方法允许进入工程着陆垫一个给定的应变轨迹的站点特定的DNA盒的整合，通过共轭ΦC31整合表达。

视频: Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

科研

生物工程

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

制造业的鲁棒天然纤维预制体的利用细菌纤维素为粘结剂

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

微生物诱导方解石沉淀通过介导<em&gt; Sporosarcina巴斯德</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

网站特定的细菌染色体工程:ΦC​​31整合介导的盒式交换（IMCE）

网站特定的细菌染色体工程:ΦC31整合介导的盒式交换（IMCE）