... you could obtain arrays A, B, C, AB, AC, BC, and ABC). Nielsen Lab E. coli CRMAGE Plasmids: The CRMAGE...Multiplex Transcriptional Repression in E. coli.” ACS Synthetic Biology 4(9) (2015):987-1000. PubMed PMID...nontargeting spacer flanked by two CRISPR repeats. The spacer can be digested using BsaI, allowing a spacer-repeat...using 3 distinct spacer-repeats, you could easily create 7 unique arrays (e.g. for spacers A, B, and C, you...promoter-gRNA construct, but with different sequences adjacent to the sites. When digested with the appropriate...plasmids are digested, unique overhangs (here, O1-4) adjacent to the cut sites “link” fragments together and...

...two-component systems. Schmidl SR, Sheth RU, Wu A, Tabor JJ. ACS Synth Biol. 2014 Nov 21;3(11):820-31. doi: 10.1021...

...PMC3785459. 2. Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher DS, Maratos-Flier E, Roth BL, Lowell BB. ...

... modular assembly of broad-host-range plasmids." ACS synthetic biology 7.5 (2018): 1279-1290. PubMed PMID...

...AsLOV2 protein binder plasmids here! Reis JM, et al. ACS Synthetic Biology. 2018 PubMed PMID: 30203962. ...

...in more contexts. References Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR...developed the Phage-Assisted Continuous Evolution (PACE) and Phage-Assisted Non-Continuous Evolution (PANCE...mutations, selection, and replications per day.  The PACE and PANCE systems use M13 bacteriophage, which rely...dilution step represents the major difference between PACE and PANCE: for PANCE the phage progeny is not diluted...-stringency PANCE, followed by higher stringency PACE, resulted in the evolution of TadA variants with...

...Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol 4:723–728 . https://doi.org/10.1021/sb500351f...for Modular Assembly of Broad-Host-Range Plasmids. ACS Synth Biol 7:1279–1290 . https://doi.org/10.1021/... most CRISPR-based genome engineering has taken place in other organisms. In many bacteria, unlike other...and chloramphenicol resistance pCRISPR: carries a spacer targeting the gene of interest and kanamycin resistance...template, and the locus cannot be recognized by the spacer-derived crRNA. However, if recombineering is unsuccessful...survive have inactivating mutations in the Cas9 or spacer sequence, and these rare events are easily detectable...successfully in Tatumella citrea, another species of Enterobacteriaceae, without the need for modification. This ...

...provided by the Rinehart lab. Steinfeld et al., ACS Chem Biol 2014 May 16;9(5):1104-12. Heinemann et... your own designer bacteria. St-Pierre et al., ACS Synth Biol 2013 Sep 20;2(9):537-41. DREADD-based... One. 2011. Feb 18;6(2):e16765. Engler et al., ACS Synth Biol 2014. Feb 5 (Web); DOI: 10.1021/sb4001504...endogenous protein of interest and co-expressing its replacement recombinant protein with a FKBP domain and a ...tyrosine kinases (RTKs) are a large class of cell-surface receptors which play a critical role in development...dimerize on exposure to light, bringing the intracellular kinase domains into contact and initiating signaling...optimal scaffold can depend on the length of the spacer region between the target sequences. These updates...

...retain some ability to induce Cas9 to make edits (AC, WLC, and JQ, unpublished results). An additional...that is complementary to the target site, or protospacer, in the genome. Native Cas9 has two catalytic...which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates...

...plasmids from Aranda-Díaz et al. Aranda-Díaz, et al. ACS Synth Biol. 2017. Pubmed PMID: 27930885. ...or noise, in reporter expression. To do so, one places reporter expression under the control of two separate...

...Wingler L, Pascolutti R, Paulo JA, Gygi SP, Kruse AC (2017) Multidimensional Tracking of GPCR Signaling...is initiated when a ligand binds to the extracellular surface of the GPCR. This results in a conformational...induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand dissociation...coupled with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G ...relatively insensitive to their endogenous ligand, acetylcholine, and are only activated upon binding of CNO ...

... the decaffeination plasmid here. Quandt et al. ACS Synth Biol. 2013. PubMed PMID: 23654268. FLINC...OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original version containing...substrate, inducing a conformational change that placesTagRFP-T and Dronpa in close proximity. By monitoring...

...Receptors Exclusively Activated by Designer Drugs. ACS Chemical Neuroscience 6:476–484 . https://doi.org...nonnative ligands. However, they retain their intracellular interactions and still set off the same pathways...

...editing toolbox.  References Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR...in the genomic DNA to contain a specific protospacer adjacent motif (PAM) made up of 5′-NGG-3′ (“N” can...eighth round of ABE evolution and the “x” is the placeholder for the number that identifies the mutation in...to ABE7.10. Depending on the position in the protospacer, the editing capacity of the ABE8s ranged from...To expand the targeting scope of ABE8, the team replaced S. pyogenes Cas9 in the ABE complex with PAM-variant...

...Jones, Nicola J. Patron, and Sylvestre Marillonnet. ACS Synth. Biol., Just Accepted Manuscript, DOI: 10.1021...

...Ronald T. Raines. "Bright ideas for chemical biology."ACS chemical biology 3.3 (2008): 142-155. PubMed PMID...from ensemble imaging to single-molecule imaging places a greater burden on the photon budget, which determines...

...H3 me1, me3, and ac – denoting mono-methylation, tri-methylation, and aceylation, respectively (these...a genomic region that has mono-methylated and acetylated histones, but not tri-methylated histones, functions...denote enhancers as follows: (mono_methylation + acetylation) - tri_methylation In the next section we’ll ...interests span machine learning, proteomics, and user interfaces for math.     Additional Resources Beyond the...

...References and resources References Bertiaux E, Balestra AC, Bournonville L, et al (2021) Expansion microscopy...?  ExM provides a straightforward answer to the space problem. Instead of imaging smaller, what if we ...

...Integration Using CRISPR-Associated Transposases. ACS Synth Biol 9:1998–2008 . https://doi.org/10.1021/...top):  A CRISPR RNA (crRNA) comprising a 32 nt spacer sequence specifying the target site. Four proteins...INTEGRATE using a single plasmid containing a multi-spacer CRISPR array. Workflow and technical considerations...INTEGRATE system follows a simple workflow: Clone spacer(s) into the CRISPR array Clone desired DNA cargo...

...Integration Using CRISPR-Associated Transposases. ACS Synth Biol 9:1998–2008. https://doi.org/10.1021/acssynbio...the cell. The edit in the chromosome can now take place. After editing, pTargetF can be cured from the cell... E. coli with the donor DNA for editing to take place. Then plasmids are cured from the cell. Steps 1-...involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed...counterselection to yield pEcCas. In pTargetF, we replaced the original 20 bp specific sequences on pTargetF...serves as a counterselection marker and will be replaced by the sgRNA targeting the genomic site of interest... clearance. It is more convenient to update the spacer(20nt) on pEcgRNA plasmid as only two 24 nt oligos...