...with a single insertion because it will segregate 3:1 in the T2 generation. You also have no control over... transgenic plants in the process.     References 1. Emami, S., Yee, M. and Dinneny, J. R. (2013) ‘A robust...transformation’, Plant Methods. BioMed Central, 2(1), p. 19. doi: 10.1186/1746-4811-2-19. PubMed PMID: 17087829..., a graduate student at Stanford University. Arabidopsis is a fantastic model organism for many reasons... are many motivations to generate transgenic Arabidopsis, from studying transcriptional and translational... plants, to complementing mutant phenotypes. Arabidopsis is amenable to the floral drip or dip transformation...transformation protocol, as well as protocols for growing Arabidopsis are also available (Weigel and Glazebrook, 2002...

... expression F- ompT lon hsdSB (rB- mB-) gal dcm araB::T7RNAP-tetA General protein expression  BLR ...folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common to all or most expression...curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains are...recommended for toxic proteins BL21-A1 Tetracycline Arabinose-inducible expression of T7 RNAP; IPTG may still...expression strain to make sure they are compatible. An arabinose-inducible plasmid will not express in an IPTG ...

...a wide range of inducer concentrations (0.1-1% L-arabinose) without signs of toxicity or burden relative...bacteria have colonized (Fig. 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram...ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate expression...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ... tumors and tumor-homing bacteria. Nat Biotechnol 1–13 (2023) doi:10.1038/s41587-022-01581-y  ...for longer. Cultures induced with arabinose and colonies on arabinose-containing plates will be visibly... inducible expression systems in E. coli. The arabinose-inducible promoter pBAD and a low-copy number ...

...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... be collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore... (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional resources on the Addgene blog  CRISPR 101...occurs in T0 plants (from callus) or T1 plants (Arabidopsis floral dip). In the next generation the CRISPR...planning to use it to simplify my work with an Arabidopsis mutant that can’t set seeds. I study the development...   Fig. 3: (Top right) A RUBY-expressing Arabidopsis seed following floral dip transformation next...without antibiotics. (Top left) Mature silique (Arabidopsis fruit) with fertilized developing seeds from ...

...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...amber (UAG) stop codon by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF...) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning and...strain for cloning repetitive DNA. endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36...of an E. coli K-12 strain. F- λ- ilvG- rfb-50 rph-1 NEB Stable   For cloning into and storage of lentiviral...

...oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect candidate for introductory...Transport, biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these...with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899-x Yokoyama...is as a biomedical model organism.  Arabidopsis thaliana Arabidopsis thaliana is one of, if not the most...community.  The Arabidopsis genome project marked the beginning of widespread Arabidopsis genetics research...characterized genome, and widespread utilization, Arabidopsis has made significant contributions to understanding...understanding plant and crop science alike.  Arabidopsis has many of the traits that make an ideal model organism...

... that dimerizes upon exposure to blue light (Fig. 1). Another blue light-inducible dimerization domain...expression needed for most applications.   Figure 1: With blue light, the VVD domains of the BLADE dimerize...AraC system, which uses arabinose, requires many wash steps to remove arabinose making it difficult to ...🎶 “Bye bye L-arabinose drivePut your cultures in the shakerTurn the LEDs onAnd when you wantYou can ...recently published in @nchembio Title: Bye bye L-arabinose drive. Dedicated to Edo, Armin and @KhammashLab...lab,” says Di Ventura. It relies on the sugar arabinose to activate transcription by causing conformation...to regulate quickly. Arabinose also circulates within growth media and can’t be contained to a specific...

...where she focuses on (1) salt-induced changes in root-to-shoot ratio in Arabidopsis, (2) expression patterns...the journal-specific guidelines.  Peer-review step 1: The first read At the first read of the paper, I ... of Arabidopsis HapMap population in Amsterdam. Currently, she is a postdoc at KAUST, Saudi Arabia, in...Julkowska, a postdoctoral researcher at KAUST, Saudi Arabia. From the perspective of an author submitting ... specific repositories (Addgene, WormBase, Arabidopsis.info). These repositories will ensure that the ...guest blogger Magdalena Julkowska from KAUST, Saudi Arabia. Magdalena Julkowska did her Ph.D. on salt-stress...

...General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein...and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible...Description Article PI Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling...Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) for use with mNG2(11) Improved yellow-...sfCherry2 sfCherry2(1-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split...brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular Fluorescence...even from a different base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles...

...dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of...in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing effect...modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be simultaneously...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...amiRNAs based upon miRNA precursors of rice and Arabidopsis species, respectively. Short hairpin RNA (shRNA...

...new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced pU6cm...amplification of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-NC and...conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling..., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation... to drive prime editing in both tomatoes and Arabidopsis, suggesting the improvements may be broadly applicable...enables heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https...

...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements...used for bacterial protein purification. When arabinose is absent, regulatory protein AraC binds O and...pBad, blocking transcription. The addition of arabinose causes AraC to bind I1 and I2 sites, allowing ...allowing transcription to begin. In addition to arabinose, cAMP complexed with cAMP activator protein (CAP) can...

... medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research is to enhance ...activities in seed plants." Plant cell reports 9.1 (1990): 1-5. PubMed PMID: 24226366. Lee, Min Woo, and Yinong...   C. roseus is not a model organism such as Arabidopsis or tobacco. Studying gene function in C. roseus...method for overexpressing genes In plants like Arabidopsis and tobacco, Agrobacterium infiltration into ...expression assay by agroinfiltration of leaves." Arabidopsis Protocols. Humana Press, 2006. 225-229. PubMed...transient gene expression analysis in seedlings of Arabidopsis and other plant species." Plant methods 5.1 (...of our plant biology blog posts Find tips for Arabidopsis transformation Read our Plasmids 101 blog posts...

... requires selection. These two steps are (Figure 1): The initial plasmid integration step (the so-called...antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover”) which leads... loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker...Current protocols in molecular biology 106.1 (2014): 1-16. PubMed PMID: 24733238. Additional resources on...tested their expression under both rhamnose- and arabinose-inducible promoters. Tight control of toxin expression...though Khetrapal et al. also demonstrated that the arabinose promoter worked too. Rhamnose induction relies...

...Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 ...collection! A “complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas...the brunt of the mechanical work for editing. Class 1 CRISPR systems make up the majority of CRISPR-based...CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different types, with ...making up type I, the most abundant type in the Class 1 category. These type Is can then be further broken...editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13226-x Cameron...been challenging. It can be difficult to ensure comparable expression of all components of Cascade for maximum...

...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...from the 5’ end and generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system...and tags.  The typical frequency of recombinants is 1 positive clone out of 104 to 105 colonies. Pro Tip...dsDNA with a recombination frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing...replication. There are two ways to avoid activating MMR: 1) use a strain of bacteria that has key MMR proteins...or 2) specially design ssDNA oligos to avoid MMR: 1) E. coli with inactivated MMR: Using E. coli with ...

...recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...an RSM is to sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies...in the Materials and Methods section of our paper (1). Lastly, it is possible to build gene regulatory ... not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids (...inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register...performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160...in the Material and methods section of our paper (1). How might one troubleshoot GSRMs that don't work...

... Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et...coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites... update the pCas/pTargetF system.   Figure 1: The editing process begins with the addition of the...updates involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2)...biosynthesis, we constructed a strain library with 1-11 copies of integrated genes. We obtained a stable...temperature-sensitive replicon for self-curing, an arabinose inducible lambda-red gene, and an IPTG-inducible...

...deletions, point mutations PCR Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes...methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-mutagenesis...phage recombination machinery is induced using L-arabinose. These cells can be made and frozen in large batches...

...also important for social and regulatory reasons. 1.) The transposon consists of a mirrored set of inverted...vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme required for catalysis...likely to grow in the coming decades.  References 1. Kowarz E, et al. Optimized Sleeping Beauty transposons...T cells to target CD19. Immunol Rev. 2014 Jan;257(1):181-90. doi: 10.1111/imr.12137. PubMed. Find the ...efficiencies of stable gene transfer using SB100x were comparable to stable transduction efficiencies of integrating...