Microdroplet PCR in Microfluidic Chip Based on Constant Pressure Regulation.

A device and method for the constant pressure regulation of microdroplet PCR in microfluidic chips are developed to optimize for the microdroplet movement, fragmentation, and bubble generation in microfluidic chips. In the developed device, an air source device is adopted to regulate the pressure in the chip, such that microdroplet generation and PCR amplification without bubbles can be achieved. In 3 min, the sample in 20 µL will be distributed into nearly 50,000 water-in-oil droplets exhibiting a diameter of about 87 µm, and the microdroplet will be subjected to a close arrangement in the chip without air bubbles. The device and chip are adopted to quantitatively detect human genes. As indicated by the experimental results, a good linear relationship exists between the detection signal and DNA concentration ranging from 101 to 105 copies/µL (R2 = 0.999). The microdroplet PCR devices based on constant pressure regulation chips exhibit a wide variety of advantages (e.g., achieving high pollution resistance, microdroplet fragmentation and integration avoidance, reducing human interference, and standardizing results). Thus, microdroplet PCR devices based on constant pressure regulation chips have promising applications for nucleic acid quantification.

Constant Pressure-Regulated Microdroplet Polymerase Chain Reaction in Microfluid Chips: A Methodological Study.

Digital polymerase chain reaction (PCR) technology in microfluidic systems often results in bubble formation post-amplification, leading to microdroplet fragmentation and compromised detection accuracy. To solve this issue, this study introduces a method based on the constant pressure regulation of microdroplets during PCR within microfluidic chips. An ideal pressure reference value for continuous pressure control was produced by examining air solubility in water at various pressures and temperatures as well as modeling air saturation solubility against pressure for various temperature scenarios. Employing a high-efficiency constant pressure device facilitates precise modulation of the microfluidic chip's inlet and outlet pressure. This ensures that air solubility remains unsaturated during PCR amplification, preventing bubble precipitation and maintaining microdroplet integrity. The device and chip were subsequently utilized for quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene, with results indicating a strong linear relationship between detection signal and DNA concentration within a range of 101-105 copies/µL (R2 = 0.999). By thwarting bubble generation during PCR process, the constant pressure methodology enhances microdroplet stability and PCR efficiency, underscoring its significant potential for nucleic acid quantification and trace detection.

Quantitative detection of urinary bladder cancer antigen via peptide-immobilized magnetic bead-based SERS probe.

Antibody pairing is a difficult step in developing all immune-sandwich assay for antigen detection. Urinary bladder cancer (UBC) antigen is a typical bladder cancer biomarker for the early diagnosis of bladder cancer. Based on peptide-antibody pairing, a surface-enhanced Raman scattering platform for the ultrasensitive detection of UBC is presented. The phage display tech was used to screen and obtain a 12-peptide ligand against UBC (KD = 4.84 × 10-7 M). Twelve-peptide-conjugate magnetic beads (MNs@12-peptide) and antibody-conjugate silver nanoparticles (AgNPs@Ab) were prepared for SERS measurements. AgNPs@Ab can be linked onto the surface of MNs@12-peptide through ligand/antibody recognition to assess a sandwich-shape complex, which turns on the SERS signal of 4-ABP. Furthermore, the second SERS signal amplification is from the magnetic field-induced spontaneous collection effect. The above design enhances the SERS signal to achieve the limit of detection as 6.25 ng/mL, the clinical threshold of 10 ng/mL. Six clinical urine samples from bladder cancer patients and healthy volunteers were also successfully detected using the dual enhancement SERS measurement. The proposed method provides the future direction of fully automated and ultrasensitive assays.

Cloning and identification of a new repressor of 3,17ß-Hydroxysteroid dehydrogenase of Comamonas testosteroni.

BACKGROUND: 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17ß-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17ß-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17ß-HSD are still unknown. METHODS AND RESULTS: In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17ß-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17ß-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17ß-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17ß-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction. CONCLUSIONS: The second tetR gene acts as a negative regulator in 3,17ß-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.

Numerical Simulation and Experimental Verification of Droplet Generation in Microfluidic Digital PCR Chip.

The generation of droplets is one of the most critical steps in the droplet digital polymerase chain reaction (ddPCR) procedure. In this study, the mechanism of droplet formation in microchannel structure and factors affecting droplet formation were studied. The physical field of laminar two-phase flow level was used to simulate the process of droplet generation through microfluidic technology. The effect of the parameters including flow rate, surface tension, and viscosity on the generated droplet size were evaluated by the simulation. After that, the microfluidic chip that has the same dimension as the simulation was then, fabricated and evaluated. The chip was made by conventional SU-8 photolithography and injection molding. The accuracy of the simulation was validated by comparing the generated droplets in the real scenario with the simulation result. The relative error (RE) between experimentally measured droplet diameter and simulation results under different flow rate, viscosity, surface tension and contact angle was found less than 3.5%, 1.8%, 1.4%, and 1.2%, respectively. Besides, the coefficient of variation (CV) of the droplet diameter was less than 1%, which indicates the experimental droplet generation was of high stability and reliability. This study provides not only fundamental information for the design and experiment of droplet generation by microfluidic technology but also a reliable and efficient investigation method in the ddPCR field.

Multiple Bio-Inspired Father-Son Underwater Robot for Underwater Target Object Acquisition and Identification.

Underwater target acquisition and identification performed by manipulators having broad application prospects and value in the field of marine development. Conventional manipulators are too heavy to be used for small target objects and unsuitable for shallow sea working. In this paper, a bio-inspired Father-Son Underwater Robot System (FURS) is designed for underwater target object image acquisition and identification. Our spherical underwater robot (SUR), as the father underwater robot of the FURS, has the ability of strong dynamic balance and good maneuverability, can realize approach the target area quickly, and then cruise and surround the target object. A coiling mechanism was installed on SUR for the recycling and release of the son underwater robot. A Salamandra-inspired son underwater robot is used as the manipulator of the FURS, which is connected to the spherical underwater robot by a tether. The son underwater robot has multiple degrees of freedom and realizes both swimming and walking movement modes. The son underwater robot can move to underwater target objects. The vision system is installed to enable the FURS to acquire the image information of the target object with the aid of the camera, and also to identify the target object. Finally, verification experiments are conducted in an indoor water tank and outdoor swimming pool conditions to verify the effectiveness of the proposed in this paper.

Characterization of a LuxR repressor for 3,17ß-HSD in Comamonas testosteroni ATCC11996.

3,17ß-Hydroxysteroid dehydrogenase in Comamonas testosteroni (C. testosteroni) is a key enzyme involved in the degradation of steroid compounds. Recently, we found that LuxR is a negative regulator in the expression of the 3,17ß-HSD gene. In the present work, we cultured wild-type and LuxR knock-out mutants of C. testosteroni with inducers such as testosterone, estradiol, progesterone or estrone. HPLC analysis showed that the degradation activities towards testosterone, estradiol, progesterone, and estrone by C.T.-LuxR-KO1 were increased by 7.1%, 9.7%, 11.9% and 3.1%, respectively compared to the wild-type strain. Protein conformation of LuxR was predicted by Phyre 2 Server software, where the N-terminal 86(Ile), 116(Ile), 118(Met) and 149(Phe) residues form a testosterone binding hydrophobic pore, while the C-terminus forms the DNA binding site (HTH). Further, luxr point mutant plasmids were prepared by PCR and co-transformed with pUC3.2-4 into E. coli HB101. ELISA was used to determine 3,17ß-HSD expression after testosterone induction. Compared to wild-type luxr, 3,17ß-HSD expression in mutants of I86T, I116T, M118T and F149S were decreased. The result indicates that testosterone lost its capability to bind to LuxR after the four amino acid residues had been exchanged. No significant changes of 3,17ß-HSD expression were found in K354I and Y356 N mutants compared to wild-type luxr, which indicates that these two amino acid residues in LuxR might relate to DNA binding. Native LuxR protein was prepared from inclusion bodies using sodium lauroylsarcosinate. Molecular interaction experiments showed that LuxR protein binds to a nucleotide sequence which locates 87 bp upstream of the ßhsd promoter. Our results revealed that steroid induction of 3,17ß-HSD in C. testosteroni in fact appears to be a de-repression, where testosterone prevents the LuxR regulator protein binding to the 3,17ß-HSD promoter domain.

Digital PCR is a sensitive new technique for SARS-CoV-2 detection in clinical applications.

The global coronavirus disease 2019 (COVID-19) pandemic has posed great challenges in people's daily lives. Highly sensitive laboratory techniques played a critical role in clinical COVID-19 diagnosis and management. In this study the feasibility of using a new digital PCR-based detection assay for clinical COVID-19 diagnosis was investigated by comparing its performance with that of RT-PCR. Clinical patient samples and samples obtained from potentially contaminated environments were analyzed. The study included 10 patients with confirmed COVID-19 diagnoses, 32 validated samples of various types derived from different clinical timepoints and sites, and 148 environmentally derived samples. SARS-CoV-2 nucleic acids were more readily detected in respiratory tract samples (35.0%). In analyses of environmentally derived samples, the positivity rate of air samples was higher than that of surface samples, probably due to differences in virus concentrations. Digital PCR detected SARS-CoV-2 in several samples that had previously been deemed negative, including 3 patient-derived samples and 5 environmentally derived samples. In this study digital PCR exhibited higher sensitivity than conventional RT-PCR, suggesting that it may be a useful new method for clinical SARS-CoV-2 detection. Improvement of SARS-CoV-2 detection would substantially reduce the rates of false-negative COVID-19 test results, in particular those pertaining to asymptomatic carriers.

The characterization of a short chain dehydrogenase/reductase (SDRx) in Comamonas testosteroni.

C. testosteroni is a research topic that can degrade steroid hormones into water and carbon dioxide through a series of enzymes in the body. Short-chain dehydrogenase (SDR) are a class of NAD (P) H-dependent oxidoreductases in C. testosteroni. Its main function is catalyzing the redox of the hydroxyl/ketone group of the hormone. In this paper, a SDR gene(SDRx) is cloned from C. testosteroni ATCC11996 and expressed. The polyclonal antibody was prepared and the SDRx gene knocked out by homologous recombination. Wild type and mutant C. testosteroni induced by testosterone, estradiol, estrone and estriol. The growth curves of the bacteria were measured by spectrophotometer. ELISA established the expression of SDRx protein, and high-performance liquid chromatography(HPLC) detected the contents of various hormones. The results show that the growth of wild type was faster than mutant type induced by testosterone. The concentration of SDRx is 0.318 mg/ml under testosterone induction. It has a great change in steroid hormones residue in culture medium measured by HPLC: Testosterone residue in the mutant type group was 42.4 % more than the wild type in culture medium. The same thing happens with induced by estrone. In summary, this SDRx gene involved in the degradation of testosterone and estradiol, and effects the growth of C. testosteroni.

Blood coagulation dynamic testing sensor based on electromagnetic vibration.

Rapid detection techniques and methods of blood coagulation have attracted wide attention in academia and the business community in the presence of the increased demands for rapid assessment (point-of-care testing) of patients from surgery, intensive care unit, and other departments. The differential equation of vibration system composed of elastic support and electromagnetic induction devices was set up using the principle of damping vibration and establishing the dynamics model; meanwhile, the harmonic response analysis and vibration fatigue coupling analysis were carried out, the analysis results were optimized, and the experimental device of the electromagnetic induction testing sensor was established. In addition, the experimental device with blood coagulation reagent was assorted to establish the standard point-of-care testing rapid blood coagulation detection curve, and to compare the testing curve with that of the imported point-of-care testing blood coagulation instrument. The results showed that the first-order natural frequency of the designed sensor was 102.35 Hz, the correlation between the designed sensor and the imported equipment was 0.996, and the testing repeatability of the designed sensor could reach 0.002. Therefore, the designed blood coagulation testing sensor based on electromagnetic induction had the characteristics of favorable elasticity and anti-fatigue, which could meet the accuracy requirements of clinical detection. Taken together, this study could provide the core technology for developing the point-of-care testing instrument for blood coagulation dynamic testing.

Electromagnetic induction sensor for dynamic testing of coagulation process.

With the increasing demand for coagulation POCT for patients in the surgery department or the ICU, rapid coagulation testing techniques and methods have drawn widespread attention from scholars and businessmen. This paper proposes the use of electromagnetic induction sensor probe for detection of dynamic process causing changes in the blood viscosity and density before and after coagulation based on the damped vibration principle, in order to evaluate the coagulation status. Utilizing the dynamic principle, the differential equation of vibration system comprising elastic support and electromagnetic induction device is established through sensor dynamic modeling. The structural parameters of elastic support are optimized, and the circular sheet spring is designed. Furthermore, harmonic response analysis and vibration fatigue coupling analysis are performed on the elastic support of the sensor by considering the natural frequency of the system, and the electromagnetic induction sensor testing device is set up. Using the device and coagulation reagent, the standard curve for coagulation POCT is plotted, and the blood sample application in clinical patients is established, which are methodologically compared with the imported POCT coagulation analyzer. The results show that the sensor designed in this paper has a first-order natural frequency of 11.368 Hz, which can withstand 5.295 × 102 million times of compressions and rebounds. Its correlation with the results of SONOCLOT analyzer reaches 0.996, and the reproducibility 0.002. The electromagnetic induction coagulation testing sensor designed has good elasticity and anti-fatigue, which can meet the accuracy requirement of clinical detection. This study provides the core technology for developing the electromagnetic induction POCT instrument for dynamic testing of coagulation process.

Quantitative determination of testosterone levels with biolayer interferometry.

Natural and synthetic steroid hormones are widely spread in the environment and are considered as pollutants due to their endocrine activities, even at low concentrations, which are harmful to human health. To detect steroid hormones in the environment, a novel biosensor system was developed based on the principle of biolayer interferometry. Detection is based on changes in the interference pattern of white light reflected from the surface of an optical fiber with bound biomolecules. Monitoring interactions between molecules does not require radioactive, enzymatic, or fluorescent labels. Here, 2 double-stranded DNA fragments of operator 1 (OP1) and OP2 containing 10-bp palindromic sequences in chromosomal Comamonas testosteroni DNA (ATCC11996) were surface-immobilized to streptavidin sensors. Interference changes were detected when repressor protein RepA bound the DNA sequences. DNA-protein interactions were characterized and kinetic parameters were obtained. The dissociation constants between the OP1 and OP2 DNA sequences and RepA were 9.865 × 10-9 M and 2.750 × 10-8 M, respectively. The reactions showed high specifically and affinity. Because binding of the 10-bp palindromic sequence and RepA was affected by RepA-testosterone binding, the steroid could be quantitatively determined rapidly using the biosensor system. The mechanism of the binding assay was as follows. RepA could bind both OP1 and testosterone. RepA binding to testosterone changed the protein conformation, which influenced the binding between RepA and OP1. The percentage of the signal detected negative correlation with the testosterone concentration. A standard curve was obtained, and the correlation coefficient value was approximately 0.97. We could quantitatively determine testosterone levels between 2.13 and 136.63 ng/ml. Each sample could be quantitatively detected in 17 min. These results suggested that the specific interaction between double-stranded OP1 DNA and the RepA protein could be used to rapidly and quantitatively determine environmental testosterone levels by the biolayer interferometry technique.

Functional analysis of a novel repressor LuxR in Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) ATCC11996 is a gram negative bacterium which can use steroid as a carbon and energy source. 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme for the degradation of steroid hormones in C. testosteroni. The LuxR regulation family is a group of regulatory proteins which play important role in gram negative bacterium. The luxr gene is located on 58 kb upstream of 3,17ß-HSD gene with the opposite transcription orientation in the chromosomal DNA of C. testosteroni. An open reading frame of this putative luxr gene consists of 1125 bp and is translated into a protein containing 374 amino acids. The luxr gene was cloned into plasmid pK18 and plasmid pK-LuxR1 was obtained. E. coli HB101 was co-transformed by pK-LuxR1 and pUC912-10, pUC1128-5 or pUC3.2-4 (which contain ßhsd gene and different length promoter, repeat sequences). The result of ELISA showed that LuxR protein is a negative regulator for 3,17ß-HSD expression. The luxr gene in C. testosteroni was knock-out by homologous integration. 3,17ß-HSD expression was increased in the mutant (C.T.-L-KO1) comparing to that in wild-type C. testosteroni (C.T.) after 0.5 mM testosterone induction. The mutant C.T.-L-KO1 and wild-type C. testosteroni were cultured at 27 °C and 37 °C. The result of growth curve proved that LuxR has also effect on the bacterial growth.

Phosphorescent quantum dots/ethidium bromide nanohybrids based on photoinduced electron transfer for DNA detection.

Mercaptopropionic acid-capped Mn-doped ZnS quantum dots/ethidium bromide (EB) nanohybrids were constructed for photoinduced electron transfer (PIET) and then used as a room-temperature phosphorescence (RTP) probe for DNA detection. EB could quench the RTP of Mn-doped ZnS QDs by PIET, thereby forming Mn-doped ZnS QDs/EB nanohybrids and storing RTP. Meanwhile, EB could be inserted into DNA and EB could be competitively desorbed from the surface of Mn-doped ZnS QDs by DNA, thereby releasing the RTP of Mn-doped ZnS QDs. Based on this mechanism, a RTP sensor for DNA detection was developed. Under optimal conditions, the detection limit for DNA was 0.045 mg L(-1), the relative standard deviation was 1.7%, and the method linear ranged from 0.2 to 20 mg L(-1). The proposed method was applied to biological fluids, in which satisfactory results were obtained.

Cloning and characterization of a novel ß-ketoacyl-ACP reductase from Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) is a gram negative bacterium which can use steroid as a carbon source and degrade steroid with about 20 special enzymes. Most of the enzymes are inducible enzymes. 3-Oxoacyl-ACP reductase (E.C. 1.1.1.100) alternatively known as ß-ketoacyl-ACP reductase (BKR) is involved in fatty acid syntheses. DNA sequence comparison showed that BKR belongs to the short-chain alcohol dehydrogenase (SDR) family. Our results showed that BKR is necessary for the degradation of steroid hormones in C. testosteroni. The DNA fragment of the BKR gene was cloned into an expressional plasmid pET-15b. BKR protein was expressed with 6× His-tag on the N-terminus and the enzyme was purified with Ni-column. Antibodies against BKR were prepared and a new BKR quantitative ELISA was created in our laboratory. The purified BKR is a 30.6 kDa protein on SDS-PAGE. C. testosteroni was induced by testosterone, estradiol, estriol and cholesterol. The expression of BKR was detected with an ELISA. The result showed that the BKR expression could be induced by cholesterol and estriol but not by testosterone and estradiol. BKR gene knock-out mutant (M-C.T.) was prepared by homologous integration. High performance liquid chromatography (HPLC) was used to detect steroid hormone degradation in C. testosteroni ATCC11996 and BKR gene knock-out mutant. We proved that the M-C.T. eliminated of testosterone degradation. Degradations of cholesterol and estradiol were also decreased. We conclude that the novel BKR in C. testosteroni plays an important role in steroid degradation. This work provides some new information of SDR and steroid degradation in C. testosteroni.

Characterization of 3,17ß-hydroxysteroid dehydrogenase in Comamonas testosteroni.

3,17ß-Hydroxysteroid dehydrogenases (3,17ß-HSDs) are found in all forms of life which catalyze the 3-position and 17-position reduction/oxidation of steroids. Comamonas testosteroni (C. testosteroni) ATCC11996 is a gram-negative bacterium which can use steroids as carbon and energy source. 3,17ß-HSD is an enzyme which is involved in the complete oxidative degradation of the steroid skeleton, induced in the presence of these compounds in the culture medium. Cyclizing RT-PCR (cRT-PCR) was used to investigate the transcription start site (TSS) and promoter of 3,17ß-HSD gene. To prove that 3,17ß-HSD is involved in the metabolic pathway of steroid compounds, we prepared a 3,17ß-HSD gene knock-out mutant and a mutant of C. testosteroni in which 3,17ß-HSD was expressed at high level. The results indicate that 3,17ß-HSD gene expression was induced by testosterone, but not by estradiol and cholesterol. Compared to the wild type C. testosteroni, degradation ability of testosterone and cholesterol was almost lost, and degradation of estradiol was decreased in the 3,17ß-HSD knock-out mutant. Meanwhile degradation of testosterone, cholesterol was obviously increased in the 3,17ß-HSD high expression mutant. Furthermore, the growths in the medium with testosterone, cholesterol or estradiol were impaired in 3,17ß-HSD knock-out mutant. The results showed that in addition to testosterone and estradiol, 3,17ß-HSD might be also involved in cholesterol metabolism. The location of the TSS and promoter of the 3,17ß-HSD gene were found in this work.

Cloning, expression and characterization of a putative 2,5-diketo-D-gluconic acid reductase in Comamonas testosteroni.

Aldo-keto reductases (AKRs) are a superfamily of soluble NAD(P)(H) oxidoreductases. The function of the enzymes is to reduce aldehydes and ketones into primary and secondary alcohols. We have cloned a 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene from Comamonas testosteroni (C. testosteroni) ATCC11996 (a Gram-negative bacterium which can use steroids as carbon and energy source) into plasmid pET-15b and over expressed in Escherichia coli BL21 (DE3). The protein was purified by His-tag Metal chelating affinity chromatography column. The 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene contains 1062 bp and could be translated into a protein of 353 amino acid residues. Three consensus sequences of the AKR superfamily are found as GxxxxDxAxxY, LxxxGxxxPxxGxG and LxxxxxxxxxDxxxxH. GxxxxDxAxxY is the active site, LxxxGxxxPxxGxG is the Cofactor-binding site for NAD(P)(H), LxxxxxxxxxDxxxxH is used for supporting the 3D structure. 2,5-diketo-D-gluconic acid reductase gene of C. testosteroni was knocked out and a mutant M-AKR was obtained. Compared to wild type C. testosteroni, degradations of testosterone, estradiol, oestrone and methyltestosterone in mutant M-AKR were decreased. Therefore, 2,5-diketo-D-gluconic acid reductase in C. testosteroni is involved in steroid degradation.

Cloning, expression and characterization of a putative 7alpha-hydroxysteroid dehydrogenase in Comamonas testosteroni.

The short-chain dehydrogenase/reductase (SDR) superfamily is a large and diverse group of genes with members found in all forms of life. Comamonas testosteroni (C. testosterone) ATCC11996 is a Gram-negative bacterium which can use steroids as carbon and energy source. In the present investigation, we found a novel SDR gene 7alpha-hydroxysteroid dehydrogenase (7α-HSD) which is located 11.9 kb upstream from hsdA with the same transcription orientation in the C. testosteroni genome. The open reading frame of this putative 7alpha-hydroxysteroid dehydrogenase gene consists of 771 bp and translates into a protein of 256 amino acids. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 161-165 in the 7α-HSD sequence) essential for catalytic activity of SDR proteins. To produce purified 7α-HSD protein, the 7α-HSD gene was cloned into plasmid p(ET-15b) and the over expressed protein was purified by His-tag sequence on metal chelate chromatography. To prove that 7α-HSD is involved in the metabolic pathway of steroid compounds, we constructed a 7α-HSD knock-out mutant of C. testosteroni. Compared to the wild type C. testosteroni, degradation of testosterone, estradiol and cholesterol were decreased in the 7α-HSD knock-out mutant. Furthermore, growth in the medium with testosterone, estradiol and cholesterol was impaired in 7α-HSD knock-out mutant. The results showed that 7α-HSD is involved in steroid degradation.

[Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17ß-hsd10 in mouse].

We expressed 17-hydroxysteroid dehydrogenase10 (17ß-hsd10) recombinant protein, prepared anti-17ß- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17ß-hsd10. RT-PCR was used to get the gene of 17ß-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17ß-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-ß-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17ß-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17ß-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17ß-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.

[High efficiency genome walking method for flanking sequences of cotton mitochondrial double-copy atpA gene based on optimized inverse PCR and TAIL-PCR].

Cloning of flanking sequences of double-copy gene is a challenge in molecular biology. We developed a method to solve this problem by combining an optimized inverse PCR (iPCR) with TAIL-PCR. First, Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene. Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene. Based on the obtained sequences, TAIL-PCR was performed to amplify further flanking regions of the gene. With this method, we obtained all of the EcoR I restriction fragments (2.2-5.1 kb) and Hind III restriction fragments (8.5-11.7 kb) of mitochondrial atpA gene in cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton. The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.