Bioencapsuiation of Genetically Engineered E coli Cells for Urea and Ammonia Removal

Urea and ammonia removal are needed m kidney failure, liver failure, environmental decontamination and regeneration of water supply in space travel. Standard dialysis machines are usually complex and expensive. Several alternatives have not been sufficiently effective. Prakash and Chang therefore studied the use of bioencapsulated genetically engineered E coli DH5 cells containing K. aerogens urease gene (26,27). The bioencapsuiation of the genetically engineered bacteria was prepared by the...

Elimination of False Positives

In the yeast two-hybrid screening, true positive clones exhibit reporter gene expression only when the AD library plasmid is cotransformed with the DNA-BD target plasmid. False positives are His+ or LacZ+ yeast transformant colonies that carry plasmids that do not encode hybrid proteins that directly interact. Such colonies arise in a two-hybrid system for several reasons (see ref. 57 for more information). Sometimes either the AD library or DNA-BD target plasmid alone can activate reporter...

Between Two Predetermined Protein Partners

1 Use standard methods to clone the gene encoding proteins to be tested for interaction (PI and P2) into either the pEG202 or the pJK202 vector (referred to as p-202 in the following instructions), and into the pJG4-5 vector This will allow the testing of interactions in both orientations. 2 Transform the following combinations of plasmids into EGY48 yeast, a p-202 -PI + pJG4-5-P2 + JK103 plate to ura-trp-his-b p-202 -P2 + pJG4-5-PI + JK 103 plate to ura-trp-his-c pRFHMl + pJG4-5 -P2 + JK103...

Gene therapy of Cancer

Modern molecular and biochemical techniques have made possible the isolation, amplification, and characterization of nearly any gene whose biological property is known. Our present understanding of carcinogenesis suggests that cancer has a genetic basis (2). The abnormal clonal proliferation is the result of genetic abnormalities m cells. Cloning and characterization of the genes involved m carcinogenesis have made possible the use of gene therapeutic approaches to selectively target and...

Muhammad Mukhtar Zahida Parveen and Omar Bagasra 1 Introduction

The term human gene therapy is defined as the transfer of DNA or RNA into human cells for therapeutic purposes. Significant advancements in recombinant DNA technology have led to an understanding of the molecular bases of many diseases ranging from inherited disorders to certain malignancies to infectious diseases such as acquired immunodeficiency syndrome (AIDS). The increasing ability to characterize a disease at its molecular level has made genetic interventions feasible and provides a...

Two Hybrid Library Screening

To perform a two-hybrid screening, you must introduce two fusion genes, either simultaneously or sequentially, into competent yeast reporter cells by transformation (see Table 4 Note 9). For example, you must transform competent HF7c cells with both the AD library and the DNA-BD target plasmids. Large-scale, sequential transformation (named LARGE below) is recommended if there is no selective disadvantage to cells expressing the DNA-BD target protein (i.e , the protein is not toxic to the...

Corinne E M Olesen John C Voyta and Irena Bronstein 1 Introduction

Chemiluminescent substrates have been successfully used for highly sensitive quantitation of reporter enzyme activity (see Chapters 5 and 6) and in enzyme-linked immunoassays for highly sensitive detection of many analytes (1-6). CSPD chemiluminescent 1,2-dioxetane substrate (7) for alkaline phosphatase has been used with Sapphire-II enhancing reagent for enzyme-linked immunoassay detection of chloramphenicol acetyltransferase and human growth hormone reporter gene products. These...

Alternative Yeast Two Hybrid Systems

The Interaction Trap and Interaction Mating Erica A. Goiemis and Vladimir Khazak Since the original demonstration that for proteins PI and P2 known to interact, it was possible to detect interactions between a DNA-binding domain (DBD) fused PI and activation-domain (AD) fused P2 by assaying transcriptional activation of one or more reporter genes containing cognate DNA binding site for the DBD (1), a number of groups have developed variants of this approach for purposes such as mapping...

Acquisition and Stability of GFP Fluorescence 1 1 31 Photobleaching

The GFP fluorescence is very stable in a fluorometer (Ward, W. W., personal communication). Even under the higher intensity illumination of a fluorescence microscope, GFP is more resistant to photobleaching than is fluorescein (25). The fluorescence of wt GFP, GFP-S65T, and RSGFP is quite stable when illuminated with 450-490 nm light (the major excitation peak for GFP-S65T and RSGFP, but the minor peak for wt GFP). Some photobleaching occurs when wt GFP is illuminated near its major excitation...

Purification and Preparation of Antibodies for Coupling to Activated Resin

1 Swell 1 5 g of protein A-Sepharose CL4B at a ratio of 1.14 (w v) in TEN buffer for at least 1 h at room temperature Wash the swollen resin (6 mL vol) twice in TEN buffer by gentle mixing Remove each wash by centrifiigation at 1500g for 5 mm Resuspend the washed resin in 10 vol of TEN buffer and pour into a 1.5 x 20 cm Econo column connected to a peristaltic pump located in line postcolumn Pack the column at a flowrate of 0.5 mL min Pack a 1-mL precolumn with SepharoseCL-4B in TEN buffer in a...

In an Immunoisolation Device for Gene Therapy

Geller, Tracy J. Thomas, Daniel R. Boggs, Susan K. Young, JoAnne Crudele, Laura A. Martinson, David A. Maryanov, Robert C. Johnson, and James H. Brauker Treatment of genetic deficiency diseases, the so-called inborn errors of metabolism has been fairly limited in scope. Whereas there has been significant success with injections of the missing gene product in cases such as diabetes and hemophilia, management of most genetic diseases has been limited to...

Luigi Catanzariti and Brian A Hemmings 1 Introduction

In renal epithelial cells LLC-PK, (I) expression of the urokinase-type plasminogen activator (uPA) gene is strongly induced by first messengers that stimulate intracellular cAMP synthesis (2). The increase of cAMP leads to a 200-fold activation of uPA gene transcription (4). In response to optimal concentrations of the peptide hormone calcitonin, i e., for which the cells express a Gs-coupled receptor (3,9), high levels of uPA protein are synthesized and secreted. The uPA protein is a serine...

Membrane Bound Alkaline Phosphatase AP as a Marker of Retroviral Infection

Moloney murine leukemia virus (MoMuLV) (7), Rous sarcoma virus (RSV) (3), and HIV-1 (8) vectors that harbor the native membrane bound form of hPLAP have all been developed. Typical applications of these vectors would include the analysis of viral infection (for example, for quantitating the efficiency of viral receptor-virion Env glycoprotein interactions refer to Rong and Bates, 1995 2 ) and cell lineage mapping (3,7). An experiment that uti- Fig. 2. Demonstration ofSEAP as a reporter gene...

Histochemical Detection of 3Gal in Fixed Specimens

In histochemical applications, X-gal is the most popular P-gal substrate. When cleaved by P-gal, it forms a blue precipitate that is robust to subsequent sectioning and histological examination. The precipitate is electron dense and thus the staining can be examined by electron microscopy (34). Other chromogenic substrates have also become available and can be used m instances in which different colors are desirable. For example, the substrate red-gal Research Organics) forms a red precipitate...

Vector Construction and Polymer Gene Expression

Concatemer genes recovered by the above procedure, including the (GVGVP)121, were ultimately placed into the expression vector pET-1 Id (Novagen, Madison, WI) immediately adjacent to the initiator ATG codon, This vector is part of the T7 expression system (11) that utilizes the coliphage T7 RNA polymerase to drive expression from the T7 promoter on the plasmid. In this case, the polymerase is provided by the host strain HMS 174 (DE3), a lambda lysogen carrying the T7 RNA polymerase gene on the...

Introduction

The yeast two-hybrid system is a powerful in vivo method for identifying novel genes encoding proteins that interact with a protein of interest 1,2 The system offers several innovations for identifying interacting proteins over conventional biochemical methods such as coimmunoprecipitation and affinity copurification. The two-hybrid system is the first genetic and molecular approach to detect interacting proteins, and the assay is performed in vivo rather than in vitro, which allows detection...

Detection of iGal Activity in Living Cells

We will consider the fluorescent substrates that can be used to stain living P-gal expressing cells. FDG fluorescein di-P-d-galactopyranoside was the first of these various fluorescent substrates to be developed, and it has proven useful as a vital stain for the identification of p-gal positive cells for subsequent cloning. FDG is a very sensitive P-gal substrate due to the sensitivity of fluorescence detection 35 for further information, see catalog of Molecular Probes, Eugene, OR . The use of...

Yeast Growth and Maintenance

YPD medium. 20 g L Difco peptone, 10 g L yeast extract, 20 g L agar for plates only Add H20 to 950 mL. Adjust pH to 5 8, autoclave, and cool to -55 . Add dextrose glucose to 2 50 mL of a sterile, 40 stock solution per liter 2. SD medium see Note 3 6.7 g L Difco yeast nitrogen base without amino acids Difco , 20 g L agar for solid medium only Add H20 to 850 mL L, add 100 mL of the appropriate 10X dropout solution, adjust to pH 5 8, autoclave, and cool to 55 C Then add 50 mL of a sterile, 40...

Preparation of CSPD Chemiluminescent Substrate Solution

The CSPD Chemiluminescent substrate is provided in the Great EscAPe kit as a 25 mM stock solution. This solution should be stored in the dark at 4 C, and is stable in this form for 6 mo. A final CSPD concentration of 1.25 mM is recommended for chemiluminescent detection of SEAP activity 20X dilution of CSPD stock solution . The stock CSPD is diluted with a chemiluminescent enhancer solution provided as a component of the Great EscAPe kit. The dilution with enhancer should be performed just...

Direct Incorporation of Nonradioactive Labeled Nucleotides

Several nonradioactive labeled nucleotides are available from various sources i.e., dCTP-Biotm, digoxin II-dUTP, etc. . These nucleotides can be used to directly label amplification products, and then, the proper secondary agents and chromogens can be used to detect the directly-labeled in situ amplification products see Section 2., steps 1 and 12 . However, in our opinion as well as in the opinions of several other laboratory groups the greatest specificity is only achieved by conducting...

Expression of GAL4 in E coli

1 LB-Medium 5 g yeast extract, 10 g bactotrypton, 5 g NaCl L of medium, autoclave. 2 Antibiotics. 1000X stock solution of ampicilhn 50 mg dissolved in 1 mL H20, sterilize by filtration and store at -20 C 1000X stock solution of kanamycin-sulfate 25 mg dissolved in 1 mL H20, sterilize by filtration and store at 20 C . 3. IPTG Prepare a 1M stock solution by dissolving 11 9 g of isopropyl-P-D-thiogalactopyranoside IPTG in 50 mL of H20. Aliquots thereof should be stored 4 Bacterial strains and...

Fluorescence Activated Cell Sorting FACS

Several investigators have sorted GFP expressing cells by flow cytometry 38,63,64 . In particular, good results have been obtained in FACS analysis with plant protoplasts, yeast, E. coli, and mammalian cells infected with bacteria expressing GFP. Fig, 3. Excitation dashed lines and emission solid lines spectra of wt GFP gray lines and GFP-S65T black lines Although precise data are not available, the excitation and emission spectra of RSGFP appear to be very similar to those of GFP-S65T 21,...

Human Placental Alkaline Phosphatase as a Marker for Gene Expression

Introduction Much of current biomedical research requires that the expression of a gene or DNA sequence under investigation be detected by rapid and reliable means. To achieve this, it is frequently convenient to place a marker or surrogate gene under the genetic control of the regulatory sequences of interest. Expression of the marker gene is then monitored by analyzing accumulation of the encoded protein and this, in turn, serves as a measure of gene...

Separating the Protein of Interest from MBP after Factor Xa Cleavage

Method I DEAE- or Q-Sepharose Ion Exchange Chromatography This method not only purifies the target protein away from MBP and factor Xa, but also provides an additional purification step for removing trace contaminants. A disadvantage is that occasionally the peak containing the protein of interest overlaps with MBP or factor Xa, giving poor separation see Note 19 The procedure is written for lt 25 mg, and can be scaled up for larger amounts. 1 Dialyze the fusion protein cleavage mixture...

SEAP Reporter Vectors

Alkaline Phosphatase Diagram

PSEAP-Basic lacks eukaryotic promoter and enhancer sequences and has a multiple cloning site MCS that allows promoter DNA fragments to be inserted upstream of the SEAP gene. Enhancers can be cloned into either the MCS or unique downstream sites. The SEAP coding sequences are followed by an intron and polyadenylation signal from SV40 to ensure proper and efficient process- Construct vector with SEAP gene under control of cis-regulatory region of interest Expression of SEAP under control of c...