Lucigen Corporation | Date: 2016-04-26
Described herein are isolated polynucleotides that encode a fluorescent protein which is at least 80% sequence identical to a protein selected from the group consisting of SEQ. ID. NOS: 2, 4, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, and 74. Also described are expression constructs containing the polynucleotides, transformed host cells containing the expression constructions, the encoded fluorescent proteins themselves, and methods of using the nucleotides and encoded fluorescent proteins for bioanalytical research.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 325.88K | Year: 2016
DESCRIPTION provided by applicant In this Phase I proposal we plan to develop two technologies to dramatically increase the expression and crystallization of properly folded recombinant membrane proteins from mammalian cell culture The most common methods to produce high yields of recombinant proteins at low cost rely on bacterial yeast or insect cell expression However the proteins produced in these systems are very difficult to purify in native form and they lack the mammalian glycosylation patterns that are often necessary for proper folding and native activity We plan to develop a novel protein fusion partner that enables mammalian expression of proteins that are otherwise nearly impossible to produce A second technology will add an internal motif to greatly enhance structural stability and facilitate crystallization of the target membrane protein Together these technologies have the potential to improve protein yields in mammalian cell culture by over fold and to allow crystallization of proteins that are currently impossible to study PUBLIC HEALTH RELEVANCE In this proposal we plan to develop technologies to vastly improve the production of properly folded mammalian membrane proteins which are critical in many human diseases This information will allow accurate characterization of the structure and activity of these proteins and it is essential for rational design of drugs to target them
Lucigen Corporation | Date: 2015-04-08
Linear vectors derived from bacteriophage of E. coli and host cells suitable for cloning are provided. The linear vectors include a left arm comprising a left telomere and a first selectable marker, a right arm comprising a right telomere and a second selectable marker and a cloning region located between the left arm and the right arm. Optional further components of the vector include transcriptional termination sequences, multiple cloning sites and reporter stuffer regions.
Lucigen Corporation | Date: 2015-06-03
Devices and methods for collecting, processing, and analyzing a sample. A sample collection module is configured for collecting, mixing diluting, and filtering a sample for analysis. A reaction cartridge is configured for processing a sample, mixing it with dried reagents, and conducting a chemical reaction for detecting target analytes.
Lucigen Corporation | Date: 2016-05-27
Methods of detecting RNA, such as ribosomal RNA (rRNA), messenger RNA (mRNA), and others. The methods include heating a cell comprising RNA in a solution to release the RNA from the cell, reverse transcribing the RNA into DNA with an enzyme, amplifying the DNA with the same enzyme, and detecting the amplified DNA. The heating, reverse-transcribing, and amplifying in at least some of the methods are performed at substantially the same temperature and a substantially constant temperature without adding additional reagents during or between the steps. The methods can be used to detect the presence of one cell type as distinguished from another cell type within a sample or to determine levels of gene expression, each without the need for elaborate extraction protocols.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 697.15K | Year: 2015
DESCRIPTION provided by applicant The human transcriptome is significantly more complex than its cognate genome due to the hundreds of thousands of possible isoforms allele specific expression issues variable RNA editing changes and differential expression patterns spanning cell types developmental stages and physiological stresses Next generation sequencing NGS platforms are fundamentally altering genetic and genomic research by providing massive amounts of data in a low cost high throughput format The main drawback of existing technologies is the short sequence read lengths they produce Illumina or the high error rate PacBio Identifying single nucleotide variations is problematic with the long read technology and de novo assembly of most transcripts is compromised with short read NGS technologies alone Even with a high quality reference human genome which is a mosaic of the parental alleles transcriptome sequencing and assembly is a significant challenge Haplotyping across an entire mRNA is critical for understanding the full extent of RNA editing and is not readily achieved without resorting to cloned DNA New tools that bridge the gap between massively parallel short read sequencing technologies and the need to assemble complete mRNA molecules are clearly needed The SBIR Phase I of this grant proposes to develop the short read NGS technology to accurately sequence mRNAs along their entire length regardless of size This technology will enable the accurate assembly of complex transcriptomes without cDNA cloning and primer walking using Sanger sequencing based strategies The development of these tools could enable the de novo sequencing of daunting transcriptomes reduce computational costs of transcriptome assembly significantly produce more complete and accurate catalogs of RNA edited transcripts and make personal transcriptome resequencing tractable PUBLIC HEALTH RELEVANCE Narrative RNA editing is a cellular mechanism that changes genomically encoded information in the expressed RNA transcripts Dysregulation of RNA editing is implicated in a number of human disorders primarily neurological and behavioral diseases We will develop new technologies to decipher where and how RNA editing events are distributed in human transcripts which regulatory sequences control the RNA editing enzymes and identify other proteins involved in editing These technologies can unlock the genetic basis of gene regulation in healthy and diseased states
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 698.42K | Year: 2015
DESCRIPTION provided by applicant A post antibiotic era of multiple drug resistance has begun to threaten the health and well being of mankind A crucial limitation in economically converting untapped natural product potential to final scalable production is the lack of next generation tools to effectively access the full medicinal impact of natural environments This project utilizes innovative technologies to capture entire small molecule pathways and express them for the first time in E coli resulting in a potent pipeline of novel environmentally derived therapeutic compounds There are two unique components available to achieve this goal A new strain of engineered E coli to support many of the unique building blocks needed for expression of natural product biosynthetic pathways and A BAC library of clones containing entire small molecule pathways up to kb to be assayed for expression of anti bacterial activities against the ESKAPE series of pathogens The outcomes of this research are expected to significantly accelerate the discovery and production of novel antimicrobial compounds PUBLIC HEALTH RELEVANCE This proposal is designed to accelerate the expression and discovery of environmentally encoded complex natural products Success will provide new antimicrobial compounds with the potential for more efficient economical rapid and effective clinical translation and large scale production
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2015
DESCRIPTION provided by applicant The human Papilloma Virus HPV causes a wide variety of diseases in humans including squamous papilloma focal epithelial hyperplasia verruca vulgaris and condyloma acuminatum In addition to benign diseases HPV infection is implicated as an etiologic agent in malignancies including cervical and anogenital cancer HPV has been identified as the most common HPV genotype associated with oropharyngeal carcinoma Morbidity mortality rates associated with these diseases can be substantial particularly when diagnosis is delayed According to recent estimates of the US population is infected with oral HPV and with HPV Annually about people in the USA are diagnosed with cancers of the oropharynx that may be caused by HPV It is projected that the number of HPV related oropharyngeal cancers will increase significantly in the future Currently HPV detection is carried out in centralized laboratories The availability of a chair side molecula test will allow dentists to detect HPV and use the test results to guide patient management Early detection of HPV related cancers will reduce morbidity mortality and cost The dentist office is a critical point of intervention for screens for HPV and other infectious agents in the oal cavity The long term goal of this project is a multiplex screening platform for HPV and other viral bacterial and fungal pathogens that go undiagnosed until they have progressed to an advanced stage and are increasingly harmful to health and more difficult to treat The isothermal amplification chemistry under development is versatile and tests for several pathogens detectable in the oral cavity are under development Providing tests that can be performed at point of care in the dentist office by a hygienist with no training in diagnostics are part of a company wide strategy to enter the molecular diagnostics market by supplying the first true point of care molecular diagnostic devices The first of Lucigenandapos s POC is due to enter clinical trials early CLIA waiver will require a level of design well beyond the typical diagnostic device to allow simplicity for use without extensive training or additional equipment for sample preparation No such CLIA waived point of care molecular devices are currently available for any disease or sample type making this proposal highly innovative Alternative POC tests are not sensitive or specific enough for reliable diagnosis Available molecular diagnostics require expensive complex instrumentation extensive training for operation and interpretation and separate sample processing Lucigen will collaborate with University of Pennsylvania Penn to develop an inexpensive automated POC device for molecular detection of HPV in saliva Lucigen will develop the isothermal DNA amplification mixes suitable for use with the Penn designed multifunctional isothermal enzymatic amplification reactor for nucleic acid detection Phase I will focus on assay and prototype breadboard development and will provide a proof of concept of the diagnostic device PUBLIC HEALTH RELEVANCE Human Papilloma virus HPV is among the most common and deadly sexually transmitted infections and is responsible for both benign genital warts and genital and oral cancers These infections are increasingly common in the oral cavity and often go undiagnosed since there is no FDA approved test for HPV in the mouth or throat Delayed diagnosis prevents early intervention that can be lifesaving The dentist office is an attractive point of testing for oral infections like HPV however this will require an HPV test suitable for use by the available personnel using the limited diagnostic equipment available in a dentist office Lucigen in collaboration with the University of Pennsylvania proposes to provide molecular diagnostic tests based on isothermal DNA amplification suitable for screening for HPV in the dentist office These tests will be simple enough for FDA clearance for use at point of care with minimal training and equipment and will be an important part of the early detection and intervention for HPV infection
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.50M | Year: 2015
Summary abstract The proteins encoded by the human genome may participate in as many as pairwise protein interactions and nearly every major process in a cell is carried out by assemblies of or more interlocking protein molecules Bruce Alberts Cell However there are no commercially available expression systems for regulated expression of multi subunit protein complexes There is an acute need for a mammalian expression system that can produce multi protein complexes is tightly regulated stable in prolonged culture and non integrated No reported expression system comes near to meeting all these needs Lucigen will develop and commercialize a stable episomal expression system with unbiased cloning ability tunable light inducible promoters improved fluorescent tags and the ability to express equivalent amounts of multiple proteins This system will facilitate direct experimental validation of the myriad protein complexes predicted by computational models as well as those that are already known e g ion channels multi subunit enzymes biochemical pathways This Phase II research will result in a novel mammalian expression vector light inducible promoters new fluorescent proteins for high throughput assays and stable cell lines that will greatly facilitate cloning expression and high throughput screening for numerous research and pharmaceutical applications The products developed in this proposal will be critical for functional analysis of the human protein interactome in normal and diseased states This knowledge will inform and confirm computational and in vitro models enabling critical advances in basic research and development of therapeutics
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2016
Expensive equipment highly trained personnel and the need for a clinical laboratory setting precludes routine nucleic acid testing NAT for infectious disease in most of the developing world and even in many resource limited parts of the United States leading to wide disparities in health care worldwide A fast sensitive low cost but a facile NAT method for robust detection of specific agents at the point of care POC would help bring molecular diagnostics to everyone The goal of this application is to demonstrate feasibility of a complete field appropriate molecular detection system suitable for use in low resource settings for the detection of three important arboviruses Chikungunya CHIKV Zika ZIKV and dengue DENV The innovative technology that is the basis of this application is a new thermostable polymerase OmniAmp which has innate reverse transcriptase RT activity OmniAmp is suitable for application in a promising NAT alternative to the polymerase chain reaction PCR called loop mediated isothermal amplification LAMP Since LAMP is isothermal it does not require specialized instrumentation plus it is much faster than PCR LAMP is also resistant to inhibitors in crude sample preparations The proposed assay will be based on LAMP using OmniAmp polymerase and performed on a simple easy to use automated molecular detection MDx platform Total assay time will be minutes with minimal hands on time and without need of any additional equipment such as pipettes centrifuge etc Results will be displayed on screen as positive or negative for a specific pathogen minimizing error s caused by user interpretation LAMP assays for detection of all major lineages of CHIKV and ZIKV including strains now circulating in the Americas and all serotypes of DENV will be developed Performance of LAMP assays will be compared to that of reference real time RT PCR methods For field use a simple and rapid sample preparation method for extraction of nucleic acid from samples whole blood and serum will also be developed In order to increase the shelf life of reagents amplification reagents will be dried down Analytical sensitivity and specificity will be evaluated by testing whole blood and serum samples by spiking them with different titers of CHIKV ZIKV and DENV serotype both individually and co infection By combining Lucigen s capacity in amplification and detection with the expertise in arboviral research and fundamental capability provided by the University of Texas Medical Branch a molecular detection system will be developed for detection and differentiation of CHKV ZIKV and DENV that can be implemented almost anywhere worldwide Successful completion of this project will lead to Development of molecular diagnostic assay for detection and differentiation of three important arboviruses CHIKV ZIKV and DENV at POC Total assay time of minutes including sample preparation and run time Detection of multiple targets in a single run from single sample prep Dried reagents for storage at ambient temperature A technology platform that can be adapted for the detection of other pathogens A simple and easy to use molecular test for detection and differentiation of arboviruses at the point of care would bring the power of molecular diagnostics to low resource settings The goal of this application is to demonstrate feasibility of a complete field appropriate molecular diagnostic system for the detection of three important arboviruses Chikungunya Zika and Dengue that can be used anywhere in the world with minimal infrastructural support and training The results of this assay will be available in minutes allowing healthcare providers to initiate appropriate actions