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...Addgene Help Center: Tech Transfer Technology Transfer Information Tech Transfer News: A Reintroduction ...Addgene account for Tech Transfer Offices. They’re packed with tech transfer resources (summaries, electronic...question? Refer them to Addgene’s Tech Transfer Team: techtransfer@addgene.org Congratulations to the Golden...stats and strategies with tech transfer offices. Drop us a line at techtransfer@addgene.org. Albert Einstein...requests from Addgene MTA: material transfer agreement Technology Transfer: the exchange of intellectual property...addgene.org identifies the hold-up. Your Material Transfer Agreement is stuck in “pending.” Fear not, there... to make MTA turnaround faster with technology transfer offices (TTOs). The latest of these efforts is...

...protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may alter protein... (78-79% after two rounds of transduction.) Compared to CCP transduction, iTOP is at least four times ..., and cell death post-transduction is low. Neuronal stem cells transduced with iTOP resulting in...lab suggests that primary cells may be better transduced using only protein. Read on for a description...description of the lab’s iTOP protein-only transduction method and its potential applications to CRISPR/Cas9 genome...genome editing.   Protein-only transduction? Niels Geijsen’s group in Utrecht wanted to pursue a nonviral...nonviral strategy for transducing primary cells. What if, instead of supplying a DNA construct, they could...

...required, through mitochondrial transfer. In the two decades since this transfer was first proposed, researchers...manipulation transplantation (AMT), CRISPR-modified adipocytes (fat cells) are transplanted as a mass that...visible chromoproteins in a cell-free transcription-translation system (Figure 6). This approach eliminates...uses a cell-free system to carry out transcription and translation of chromoprotein and gRNA plasmids. ... astrocytes Nerve-to-cancer cell mitochondrial transfer tracker Red calcium indicators PinkyCaMP and FRCaMPi...astrocytes and neurons to label surface proteins with a membrane-impermeable biotin substrate (Figure 1). Biotinylated...extracellular matrix proteins, cell adhesion molecules, transporters, ion channels, GPCRs, and more.  Figure...

... not appear to mind extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab...modular mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous...selecting the important elements from existing transposon and vector systems and creating an all-synthetic...validated this vector, called pBAM1, by conducting random mutagenesis in the soil bacterium Pseudomonas putida...family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on to characterize...mitochondrial turnover Mitochondrial health, transport and turnover are important features to be monitored...

...reagents can be delivered via transfection, nucleofection, viral transduction or injection as either protein...cell line that can be transfected at high efficiency. Lentiviral transduction Cas9 and gRNA can be present... present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene...inserted into an AAV transfer vector and used to generate AAV particles. Transient or stable expression...Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed cell lines... lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic... method: Do you need stable integration or is transient expression sufficient? Which cell types will you...

... could infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal...integration. After co-transfection of these plasmids, 293T cells released transgene-containing lentiviral...protein), Pol (polymerase), Tat (viral transactivator for transcriptional activation from the 5’ LTR) and Rev...capacity of a lentiviral vector but for which transient transgene expression is sufficient. Lentiviral vector...include a large genetic capacity and the ability to transduce both dividing and non-dividing cells. Lentiviral...integrate into the genome, permitting long-term transgene expression, but they could only infect actively...infect non-dividing cells, but without sustained transgene expression. To design a viral vector system that...

...mammalian analogue of bacterial transformation is transfection. In stable transfection, DNA from the plasmid is...vectors delivered by transformation and transfection and left the business of transduction to our Viral Vectors... Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Plasmids 101: Choosing...to retain the vector as cells divide. In transient transfection, the vector remains episomal, meaning it...vector replication at all. In these cases, transient transfection may be sufficient for your needs even without...called transfer vectors. The gene of interest is cloned into a shuttle vector for transfection into producer...has seen before, showing how DNA sequences are translated into amino acids. And I was astonished that the...

...PHP.B has a >40-fold increase in CNS transduction and transduces the majority of astrocytes and neurons... mice whose CNS was transduced by PHP.B but absent in mice who were not transduced by PHP.B. The Deverman...These results were surprising because in vivo transduction of AAV serotypes generally tracks between strains...diverse library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This... an enhanced version of PHP.B, shows similar transduction efficiency, but at a log lower dose than PHP.B...permissive and six as non-permissive to PHP.B CNS transduction. Two genes were associated with the PHP.B permissive...outbred mice (F1) had half the level of CNS PHP.B transduction as their C57BL/6J parent. The second generation...

...find the subsequent transformation protocols below: Untergasser Lab Transformation of chemically competent... easily transformed without a heat-shock step. Because of the large number of transformations being performed...that transforms well in other competent cells) to ensure that your cells can be readily transformed. Compare...Want even more tips for transforming your cells? Check out our transformation guide.   ...We perform over 100 transformations each week at Addgene, so we need to be cost and time efficient. We...a little-known reagent for streamlining the transformation step. Read on to find protocols and tips that...competent cells. If you need a high efficiency of transformation (for example, if you are working with a library...

...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically...with a large backbone might be more difficult to transform.    Figure 1: Factors to consider when ... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...contains 5,000 plasmids, your goal for your transformation would be to obtain 100 x 5,000 = 500,000 colonies...likely rely on serial dilutions to estimate transformation efficiency (Figure 2).    Figure 2: ...

...Reverse transcription of the transgene occurs in the cytoplasm, then the resulting double-stranded DNA transgene.... However, IDLV genomes are transcriptionally active, leading to transient expression of gene products...lentiviral vectors is random, and the integration site will be different for each transduced cell, there’s no...mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want your cells to ...They rely on a viral protein called reverse transcriptase to convert their RNA genomes into DNA to be ...second-generation system as an example.  The lentiviral transfer plasmid encodes your insert of interest, flanked... larger protein: PR (protease), RT (reverse transcriptase), and IN (integrase). These viral packaging ...

... the transgene of interest. In our studies, mCherry interfered with the transport to the membrane of the...the hM4Di might improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag...the expression of the transgene of interest and its localization in the membrane. The lesson we learned...2013) could help improve the transport of protein of interest the membrane.   Figure 1: Electron... the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore variants...only a small proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic...

...tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used. UAS...for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on ...This element is responsible for initiating the transcription of your insert into RNA. In practice, the term...controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role...expressed.      The RNA polymerase(s) RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria...

... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org... in 3 different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure...Figure 1: Transformation introduces transformation grade plasmids into a bacterial cell. Transformation...bacterial transformation of chemically and electrocompetent cells is minimal (picogram range). It can ...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. The amount ...without commercially available kits and DNA for transformation doesn’t need to be as pure as for other uses...

...they cannot cross cell membranes, so they are best used either with plasma membrane proteins (such as GPCRs... minimum energy transfer values you can expect, thereby establishing the dynamic range of your FRET assay...what's it good for? Fluorescence resonance energy transfer (FRET) is a biophysical interaction between two...first fluorophore (the donor) is excited, it can transfer energy directly via resonance to the second fluorophore...structural changes in the ribosome during protein translation (Ermolenko et al., 2007). All that is required...develop a successful FRET-based assay since energy transfer only occurs when donor and acceptor are close ...Å away from the donor doesn’t result in energy transfer and thus, your specific FRET signal is unaffected...

...project forward, transductions should not be rushed into. Before you start any transduction, you should always...Primers targeting the transgene are also beneficial as they ensure that the correct transfer plasmid was used... positive for vector encoded transgene expression following transduction with serial dilutions of a viral...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a ... your packaging cell line, prepped your DNA, transfected and harvested your lentivirus. Now it’s time ...assaying for expression of a gene carried on the transfer plasmid or quantifying the number of viral copies...gag, WPRE, antibiotic resistance-genes, or the transgene itself. Many researchers prefer to design primers...

... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter...lag time due to the time needed for transcription/translation. Many thanks to Nicole Zurcher who contributed... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...activator rtTA (reverse tetracycline-controlled transactivator) is normally inactive and cannot bind the tetracycline...

...packages have spent days to weeks in transit and some have been transported across oceans and continents. Each... treated to easily take in plasmids (transformation). Transformed bacteria are then grown up on antibiotics...received is immediately transformed and stored. Some samples need to be transformed with a micropulser using... performs an average of 140 transformations. The lab typically transforms newly deposited samples from...delivered by a mail courier and then the journey of transformation and storage begins. Some samples are submitted...long-term storage in our facilities, every sample is transferred to a 2D barcoded tube (see next section) and ...make a new tube from a replicate sample. The transformation After the DNA has been received and stored,...

...-visual G protein signal transducers (collectively known as the transducerome) that can be activated downstream...Resonance Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer (FRET), in which...non-visual G protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing...including smell, taste, sight, inflammation, and neurotransmission. But with approximately 950 different human...involved in sight (Olsen RHJ et al., 2020). A transducer complex consists of a trimer of three subunits...such a tool is being able to identify specific transducer complexes that are activated in response to a...fluorophore. The difference for BRET is that the energy transfer is between a luminescent donor and a fluorescent...

...undergo retrograde transport, traveling up the axon to the cell body and driving transgene expression (Tervo.... AAV8 AAV8 has high transduction efficiency in multiple tissues, rapid transgene expression, and can ...sialic acids1 AAV receptor (AAVR)2   AAV2 Heparan sulfate proteoglycan (HSPG)3 AAV receptor (AAVR...β3 AAV6 α2,3-/α2,6 N-linked sialic acids1 Heparan sulfate proteoglycan (HSPG)3 AAV receptor (AAVR...good choice for targeting neurons, with high transduction frequencies in the central nervous system (CNS...Asokan, et al., 2006). AAV1 has also been found to transduce skeletal muscle, cardiac, and retinal cells (Issa... 2006). AAV6 is particularly well-suited for transduction of lung cells via airway administration (Halbert...

...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...presence of a transgene instead, which can’t be done if your selection method kills the transgene free individual...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...For example, with CRISPR-Cas9 gene editing, transformation may be identified by resistance to an antibiotic...antibiotic. Successful editing of transformants then typically occurs in T0 plants (from callus) or T1 plants...contaminate it. I have used it for selecting transformation events and am currently planning to use it ...

...their position relative to a transcription start site. In STARR-seq, randomly sheared genomic libraries ...of these random stretches of DNA contain an enhancer, it will activate its own transcription from the ...alone. Molecular processes from signal transduction to transport rely on protein-protein interactions and...an alternative method, called STARR-seq (self-transcribing active regulatory region sequencing) that tests...single gene. The gRNA expressing cells were then transfected with a Cas9-expressing plasmid to generate a ... Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC...RAS signaling pathway and includes 180 unique transcripts. Each gene was chosen to represent the primary...

...light-dependent activity to transcription, they fused BldD to the transcriptional activators p65 and VP64 ...readily found at Addgene. Shining light on transcriptional activation using dCas9 Initial photoactivatable...early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato’s lab at ...photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65). Upon expression...the activator into position to activate gene transcription. The researchers tested a variety of combinations...CRY2FL-VP64, a fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in...(Nihongaki, et al., 2015) resulting in a double strand break (DSB) that can be repaired by either non-...

...specific titer, you can better plan your transductions and use a range of reasonable MOIs. 2. Create a monoclonal...generated from A549 cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and selected...generate DNA duplexes with one strand from the edited cells and one strand from the control cells, which.... The appearance of the 428 bp band indicates edits in the BRAF gene in the BRAF-gRNA transduced cells...generated A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting...whole-cell extracts from stable pools of A549 cells transduced with Cas9 expressing lentivirus at the indicated...

...can be motifs within transmembrane proteins, such as receptors, or post-translational modifications on those...diffuse through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear ones...Depending on the application, they can be membrane-permeant or membrane-impermeant, and the cells of interest...viability dyes are membrane-impermeant and can only enter cells with compromised plasma membranes — i.e., dead...conjugates are commercially available for a wide range of fluorophores, allowing for great flexibility ...epitope-targeting antibody can originate from a range of species; for example, rabbit or mouse. In a second...

...DNA must first be transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into...consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe, translate...expression post-transfection. Mammalian systems can express proteins both transiently or through stable...expression vector that is then transformed into a bacterial cell. Transformed cells propagate, are induced...vitro using purified components of the transcription and translation machinery. These include ribosomes, ...proteins are then modified via an array of post-translational modifications including protein folding, formation...proteins from mRNA and the addition of post-translational modifications Learn more molecular biology ...

...al., 2025). In retrotransposon insertion, a combined endonuclease-reverse transcriptase uses homology ...fused to a shortened R2Tocc transposon protein, and can "retarget" the transposon to new loci in the genome...genome. The modified transposon RNA has untranslated regions (U, blue) on the outside of the homology arms...-associated membrane protein 5 (LAMP5) — as well as cholinergic (choline acetyltransferase, ChAT) neurons...is one of the most important and abundant neurotransmitters, but it is challenging to monitor because ...rapidly taken up by postsynaptic receptors and transporters. The intensity-based Glutamate-Sensing Fluorescent...deposited versions with both PDGFR and NGR fusions for membrane display, they recommend the NGR vectors for most...

...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...expression transposases leading to increased integration frequency. (C) Increasing rounds of transfer on transposition-inducing... engineering single genome edits but the double strand breaks it introduces makes it difficult to perform...chromosomal integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these... This would result in the lack of recovered transformants. Our speculation motivated us to update the ... sequence in pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to ...

...strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Non-random integration...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...Danio rerio) have been used since the 1930’s in a range of biological studies, including investigations ... to produce abundant, externally fertilized, transparent progeny (Figure 2). These features give it a ...individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother...sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity...mutants In the 1990’s, two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants...

... a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery ...as two other potent transcriptional activators (p65 and Rta). These transcriptional activators work in ...is fused to CBP, an histone acetyltransferase domain capable of rearranging chromatin structure. This method...interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...gene regulation. This can be used to activate transcription during either initiation or elongation, depending...uses specially engineered sgRNAs to increase transcription. This is done through creating a dCas9/VP64 ...

...previously require a preliminary double stranding step. The double stranding step was assumed to be necessary...genome is single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  ...extraction, the [+] and [-] strands might naturally associate forming a double-stranded species that could be...extract is a heterogeneous mix of single-stranded and double-stranded species. While not all of the molecules...extract associate with complementary strands, enough double-stranded species exist in the mix to serve as...viral genome We hypothesized that this double stranding step might not actually be necessary. Since the...the AAV genome exists as [+] and [-] strands that are packaged at the same frequency, we reasoned that...

...), YFP (yellow), and membrane-tethered CFP (cyan). Brainbow 1.1 adds OFP (orange) (Livet et al., 2007)... mouse generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic YFP, or cytoplasmic...labeling technique involves the introduction of a transgene construct containing fluorescent proteins (XFP...Because each cell can have multiple copies of the transgene that will recombine independently, cells may acquire...respectively, cells will fluoresce either red or orange until recombination occurs. Following the development...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically...non-fluorescent form of YFP as the default state of the transgene such that recombination is necessary to excise...

...format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation...flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period... regardless of whether it interacts with a transcriptional activator (1a) or a repressor (1b). Scientists...interactions. In later years, improvements to yeast transformation protocols also helped establish the yeast two-hybrid...reporters and some that repress instead of activate transcription. The HIS3 reporter system (e.g. the Gateway-...even possible to have Y2H systems that avoid transcriptional reporters altogether; In the split-luciferase...group of prey fusions can either be generated using random genomic fragments from the organism of interest...

...cells in vitro. I transduced the cultured cells with a virus that encodes a transgenic protein for the ...having a separately expressed transduction reporter is that not every transduced cell will necessarily manage...the transgenic protein. We don't know yet how abundantly CD45 will be expressed on the transduced cells...panel to analyze transduced HEK 293T cells. Here, we looked at both the transduction efficiency and the...emission. The visible color spectrum is located in a range of wavelengths of about 380–700 nm. Photons of lower...electrons of a fluorophore, they can do so at a range of wavelengths, rather than at just one defined ...Likewise, the emitted photons also appear within a range of wavelengths. For that reason, we speak of excitation...

...HiBiT-tagged targets in translational research Researchers can incorporate HiBiT with a wide range of workflows,...fibrosis transmembrane conductance regulators (CFTR) (Kamada et al., 2023). CFTR is a membrane protein ...reporter to show that the mutants abolished transcriptional activity, even in the presence of endogenous...editors Tracking factors that utilize exosomal transport is quite challenging. Work by Shibata et al. (...converts adenosine to inosine (A-to-I) within double-stranded RNA. They hypothesized that if ADAR1 were carried...carried by extracellular vesicles (EVs), edited transcripts could endow neighboring cells with pro-oncogenic...cells proved that the enzyme was successfully transferred and internalized. Functional assays further showed...

...delivery include plasmid transfection, RNP electroporation, and viral transduction; but these methods aren...they can then fuse with the target cell membrane, and transfer their CRISPR cargo into the zygote. As ...Repair (HDR), CRISPR activation or repression, transgenic mouse line creation, and in vivo gene editing... non-replicative. Nanoblades are produced by transfecting the following four components into 293T cells...spontaneously assemble into particles at the cell membrane. Mature Nanoblades then bud into the cell culture... can also deliver Cas9 variants, such as a transcriptional activator dCas9-VPR, if these variants are ...Rless glycoprotein (BaEVRless) had high rates of transduction for most cell types they tested. Interested ...

...paternal grandmothers early food supply influenced cardiovascular mortality of the female grandchildren." BMC...histone modifications, which can influence the transcription of genes by RNA polymerase II, or by the addition...methylcytosine in CpG islands makes  genes less transcriptionally active. The role of epigenetics in studying...to play a large role in a variety of diseases, ranging from cancer, to diabetes, to congenital disorders...isolated population in Sweden found that paternal grandfathers who had experienced famine before their adolescence...adolescence passed on a heritable trait to their grandsons that decreased their likelihood of death from ...of death from diabetes. Conversely, paternal grandmothers who experienced famine while still in their ...

...genes from a single transcript, or if you want to prevent read-thru transcription from resulting in the...developing a general translation initiation sequence known as the Species-Independent Translational Sequence (SITS...institution, you can order plasmids covering a wide range of fields from Addgene. What you might not know ...extensive sequences used for cloning into the final translated products. Negative selection markers built into...multidrug efflux pump by novel and powerful pyranopyridine derivatives." Proceedings of the National Academy...3509-3514. Tikhonova, Elena B., et al. "A transcription blocker isolated from a designed repeat protein...of which you can clone your gene of interest. Translation of the standardized leader peptide prevents secondary...

...non-target strand. The helicase domain is then activated, and Cas3 begins unraveling the double-stranded DNA ...cleaving the ssDNA strands. In this way, Cas3 eats away at both the non-target and target strands by making cuts...every so often. Cutting both strands ensures that there isn’t a template strand left behind that the cell... regions on the non-target strand. Occasional cleaving of the target strand is not as well understood ...bypassed by leaving out Cas3 — a tactic some CRISPR transposase systems utilize with type I-F Cascades. ... section for details). The newly formed single-stranded DNA (ssDNA) is then pulled through Cas3. After...implies — after nicking, Cas3 reels in the non-target strand of DNA much like a fisherman would reel in a big...

...which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates...two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary to the gRNA... binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators In addition... then be fused to effector domains such as transcriptional activators or repressors. These fusion proteins...loci within a single cell. One might imagine transfecting cells with both the nuclease-positive and nuclease-null...Consequently, fewer genetic tools for targeted transcriptional and epigenetic regulation have been validated...inexpensively generate a potent Cas9-based transcriptional regulator without making any functional changes...