Research Tools : Gene expression


High-Throughput Genome Editing and Engineering of Industrial Yeast, Other Fungi

UW–Madison researchers have developed expression cassettes that facilitate genome editing and sequence replacement in fungi at an extraordinarily high rate. Their HERP (Haploid Engineering Replacement Protocol) cassettes combine thymidine kinase (TK) enzyme with meganucleases, and permit hundreds of thousands of independent transformations to be obtained in a single experiment.

TK (from human Herpes Simplex Virus) serves as both a selectable and counter-selectable marker. Since the common ancestor of all fungi lacked the gene, the marker is likely of nearly pan-fungal utility. Relevant species should include Saccharomyces cerevisiae, Saccharomyces mikatae, Saccharomyces kudriavzevii, Saccharomyces uvarum and Neurospora crassa.

Oxygen-Responsive Bacterial Gene ‘Switch’ for Biofuel Production and More

UW–Madison researchers have taken a known promoter sequence and developed several novel variants capable of modulating downstream expression levels over a 50-fold range.

The new promoters are exquisitely sensitive to changes in O2 concentration as well as other growth conditions applicable to various industrial fermentations. These include changes in the redox state of the cell and also membrane perturbations/stress caused by production or export of hydrophobic compounds, biofuel precursors or recombinant proteins.

Some of the promoters act as tightly regulated on/off gene switches while others offer a more graded or linear response.

New Protein Production Strategy for Plants

UW–Madison researchers have identified a new plant viral IRES that can facilitate the efficient expression of multiple proteins from a single mRNA. The researchers discovered the new IRES in the Triticum mosaic virus (TriMV), a wheat virus that expresses 10 proteins from a single mRNA strand.

Designing Ubiquitin Oligomers

UW–Madison researchers have developed methods for synthesizing oligomers of Ub and Ub-like polypeptides with a well-defined number of Ub units. The oligomers are constructed using thioether groups rather than the natural isopeptide linkages. The thioether groups may be designed to closely mimic the native isopeptide or may be varied.

Methods for preparing and coupling Ub building blocks (i.e., monomers) also are provided.

Bacteria Modified to Secrete Biologically Active Protein for Large-Scale Production

UW–Madison researchers have discovered E. coli mutations that substantially increase the amount of biologically active, recombinant protein secreted from cells.  The mutations disrupt genes in a YebF-mediated protein secretion pathway.  Bacteria modified to contain these mutations are useful for the production of secreted proteins.  They can be used to produce proteins that might otherwise not be expressed due to toxicity or folding errors.  They also can be used to produce secreted complexes of enzymes such as cellulases and xylanases for the manufacture of cellulosic biofuels.

Translation-Coupling Cassette for Quickly and Reliably Monitoring Protein Translation in Host Cells

UW–Madison researchers have developed a method of using translation coupling to quickly and reliably determine whether a given host is capable of expressing the gene product of any given gene. This method could be used to monitor protein translation efficiency in bacterial cells.

The method involves a cassette that couples the full translation of a desired target gene to that of a detectable response gene. If the target gene is fully translated, so is the response gene. If the target gene is not translated, the response gene product is not detectable.

For example, one embodiment utilizes a DNA plasmid with a cloning site upstream of a DNA hairpin that masks the ribosome binding site required for translation of an antibiotic resistance protein. Scientists clone the protein of interest into the plasmid without a stop codon in frame and with a tag for purification purposes. Expression of the desired protein leads to unwinding of the DNA hairpin, unmasking the ribosome binding site and promoting translation of the antibiotic resistance protein. If the protein of interest is expressed in E. coli, the bacteria will survive in the presence of the antibiotic. If the protein is not expressed, the bacteria will die when exposed to the antibiotic.

Controlling and Predicting the Stability of a Protein Against Degradation by Proteases

UW–Madison researchers have developed methods for predicting and controlling the stability of expressed polypeptides in prokaryotes, particularly mycobacteria like M. smegmatis.  They previously showed that DesA3 expressed in M. smegmatis with a modified C-terminal sequence had higher catalytic activity and stability than with the natural C-terminal sequence.  The researchers found that the identity of the last two or three amino acid residues of the C-terminus is a predictor and determinant of protein stability and resistance to proteolytic degradation. 

Specifically, altering one or more of the last three amino acid residues at the C-termini of polypeptides can make the proteins more stable during heterologous expression in mycobacterial hosts.  Identifying the last three residues also can be used to predict the relatively stability of proteins against degradation by proteases.

Recombinant oriP Plasmid Vector for Efficient DNA Synthesis

UW-Madison researchers have now improved the oriP vector by engineering the cis acting oriP element.  The recombinant vector is significantly more efficient than the wildtype vector.  It can be used to enhance stable, long term expression of a desirable gene or to express a desirable gene at higher levels.   

The origin of synthesis within oriP is the Dyad Symmetry (DS) element, which includes two pairs of EBNA-1 binding sites flanked by half-binding sites for the human protein TRF2.  The inventors discovered that the affinity of EBNA-1’s binding to an origin directly correlates with the efficiency at which DNA synthesis is initiated as well as the efficiency at which extrachromosomal establishment is supported at that origin.  They used these findings to construct artificial origins of DNA synthesis that are several-fold more efficient than wildtype DS in their abilities to initiate DNA synthesis and promote extrachromosomal establishment.

Novel Poly(A) Polymerases that Interact with Nuclear Phosphoinositide Signaling Molecules

UW-Madison researchers have identified novel PAPs that interact with phosphoinositide signaling molecules. These new PAPs, called phosphatidylinositol phosphate (PIP)-PAPs, provide a new nuclear regulatory mechanism, and therefore a new means of controlling and regulating protein expression. Unlike known PAPs, the activity of these PIP-PAPs may be directly modulated by components of phosphatidylinositol-based signaling pathways, which play crucial roles in the regulation of cell processes at the plasma membrane and in the nucleus.

The first of these PIP-PAPs to be identified is the Speckle Targeted PIP kinase alpha Regulated (Star)-PAP. Altering the expression of Star-PAP affects a set of over 300 genes, many of which encode proteins that are important in oxidative stress pathways that modulate diseases, including neuronal disease, cardiovascular disease, stroke and pulmonary disease.

Model System for Identifying Anti-Cancer Agents That Interfere with Gli2

UW-Madison researchers have developed a mutant form of Gli2 that results in a constitutively active Hh signaling pathway. They identified a point mutation that inhibits the first step in the degradation of Gli2 and makes the protein more stable.

Because the mutation increases the half-life of Gli2, it results in an increase in Gli2-dependent transcription. This in turn leads to an increase in tumorigenesis, which enables high throughput screening for potential anti-cancer compounds that interfere with Gli2-mediated tumor growth.

Compositions and Methods for Regulating mRNA Transcription and Translation

This technology provides novel nucleic acid constructs encoding the cardiovirus 2A polypeptide operably linked to a promoter. When expressed in mammalian cells, in the absence of any other viral proteins or genes, protein 2A localizes to the nucleolus, binds to ribosomes, and inhibits cap-dependent mRNA translation, effectively preventing the translation of anything that is not a viral protein. Because cardiovirus protein 2A does not inhibit cap-independent IRES-driven mRNA translation, the result is a net increase in IRES-dependent protein expression relative to cap-dependent protein expression.

A Non-Cytotoxic oriP/EBNA-1 Vector for Human Gene Therapy

UW-Madison researchers have developed a vector encoding a derivative of EBNA-1 that is not cytotoxic when expressed efficiently in cells.  The derivative lacks several amino acids from the LR1 region.  It supports extrachromosomal replication, maintenance and transcription from extrachromosomal oriP-containing vectors, but does not substantially activate expression of host cell genes. 

Expression Vector with Dual Control of Replication and Transcription

UW-Madison researchers have constructed a controlled BAC expression system that can produce large amounts of genomic polypeptide upon induction. Their modified pBAC system includes both a conditionally amplifiable origin of replication(oriV) with a broad host range, and a tightly regulated, inducible transcriptional promoter linked to a gene of interest. The conditional oriV and the inducible promoter can be activated jointly or separately by signals in the host cell.

Specifically, BAC copy number is controlled by a single inducing protein, TrfA, which induces replication from oriV. Gene transcription is controlled by an arabinose-inducible promoter, which can be induced to produce many copies of the inserted gene. Thus, this pBAC includes all of the elements necessary for transcribing large quantities of polynucleotides in a controlled fashion.

Producing Medical Toxins by Transferring Genes Between Clostridium Strains

UW–Madison researchers have developed a highly efficient conjugative gene transfer system to move toxin-producing DNA between clostridium species. The method employs a shuttle plasmid, spliced with the donor’s sequence of interest and a gene expression promoter, contained within E. coli. Strains of E. coli harboring the gene-carrying plasmids then can be cultured in close proximity to a nontoxic clostridium species, wherein copies of the plasmid will be passed into the host via conjugative transfer.

Expression and Purification of Recombinant Rabbit and Human Phosphoglucomutase

UW-Madison researchers have now developed a method for expressing and purifying PGM using an E. coli system. Their technique produces high yields of recombinant PGM in a soluble, active form. A variety of materials are available for licensing, including whole cell pastes of recombinant E. coli, cell-free extracts and purified recombinant proteins.

Promoter-Trap Plasmid for Identifying Promoters

UW-Madison researchers have developed a promoter-trap vector for use in Gram-positive bacteria such as Bacillus cereus. The promoter-trap vector was constructed to contain genes for ampicillin and chloramphenicol resistance and can replicate in E. coli and B. cereus. A multiple cloning site containing EcoRI, Sac-I, Kpn-I, SmaI, BamHI, and XbaI restriction sites was inserted at the 5’ end of a promoter-less, green fluorescent protein (GFP) marker gene. Expression of this modified GFP can be quantified by measuring fluorescence intensity and is amenable to flow cytometry and cell sorting.

Method of Expanding a Host Range of a Bacterial Plasmid

UW-Madison researchers have identified mutations in the pir gene that allow R6K to expand its bacterial host range. A single amino acid change in the pir gene allows the establishment of the R6L plasmid in phylogenetically distinct bacterial species, and also substantially increases the copy numbers.

Increasing Secondary Metabolite Production in Fungus for Drug Development

The researchers now have developed a set of genetically modified Aspergillus nidulans strains with increased secondary metabolite production. The strains overexpress one or both of the global regulators previously implicated in secondary metabolite production. Moreover, naturally occurring gene clusters in the strains are deleted to reduce competition for the desired genes.

Fusion Protein Expression Vectors, CMV-GST and CMV-tac-GST

UW-Madison researchers have developed two plasmid vectors that allow expression of the GST pi protein. These vectors were tested in bacterial and mammalian hosts, where they allowed expression of the GST protein as expected. Pieces of DNA can be inserted into a cloning site in these vectors. The encoded recombinant gene then can be expressed in bacterial and mammalian cells, where it will direct the synthesis of a fusion protein in which the amino terminal portion is a GST and the carboxy terminal end is the protein of interest.

Expression System and Fermentation Processes For Overexpression Of Holo-Acyl Carrier Protein

UW-Madison researchers have created eight bacterial expression plasmids from either E. coli or spinach, which express both apo-ACP and E. coli holo-ACP synthase, the enzyme responsible for the post-translational modification of ACP. Therefore, the plasmids allow completion of the required modification. The researchers' expression system enables the production of ACPs in high yields by using chemically defined minimal medium and specialized fermentation procedures.

A Nucleotide Element That Enhances Transcription from Bacterial Promoters

UW-Madison researchers have obtained a series of 31 sequences for the UP element, each of which enhances transcription anywhere from 136- to 326-fold over the wild type sequence, and have derived a consensus sequence from these. The sequences were chosen based on how rapidly each bound to the bacterial RNA polymerase in a cell-free assay, assuming that the kinetics of the binding reaction determined sequence affinity for RNA polymerase.

Bacterial Transcription Enhancers

UW-Madison researchers have developed a way to increase gene expression from promoters in most or all bacterial systems. More specifically, the researchers created a means to increase the rate of transcription initiation from a bacterial promoter by fusing a DNA sequence containing an alpha subunit RNA polymerase binding site to the promoter upstream of the sigma subunit of RNA polymerase binding site. The resulting DNA sequence causes RNA polymerase to bind to the promoter more efficiently, without the need for transcription factors.