Eukaryotic BioBrick Library Medical application 2/4/2019 e.g. EGFR overexpression in cancer e.g. Bax overexpression Cell stress detection systems tagging for degradation (Aptamers) upregulation of degradative machinery assay development ROS color-coded reporter (Brainbow) Ca2+ LC3-levels Eukaryotic BioBrick Library Plug-and-Play reporter library (easy to customize and combinational) Recombineering

What’s in a plasmid Promoters: Tissue specific (E.g. heart OR cancer OR disease) inducible Different efficiencies Cytomegalovirus (CMV) promoter because of its strong activity in most cell lines PolyA tail: different mRNA stabilization efficiencies (could be tested with RT-PCR) SV40 poly A tail synthetic intron enhancing mRNA stability Selection genes: 1Bacterial– AMP/ Kana Mammalian –resistance to G418, dihydrofolate reductase (DHFR), or glutamine synthetase

RNAi Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18632760

Stable integration sites (Multiple) Integration site-specific transcriptional reporter gene analysis using Flp recombinase targeted cell lines. Biotechniques. 2007 Feb;42(2):217-24. http://web.mit.edu/ccrhq/hyneslab/2nd%20floor/flip-vectors-protocols.html http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1609115 Stable acceptor cell line(s) GOI/sensor/shRNA #1 GOI/sensor/shRNA #2 Bidirectional/multiple CDS…. (high complexity)

Site-specific recombination Useful for ON/OFF Proteins of interest - shRNA Cre/lox system Splicing/recombination catalyzed by Cre recombinase LoxP, binding sites for Cre, have directionality KI, KO, inversion possible FLP-FRT system FLP recombinase Active at FRT (FLP recombinase target) sites inversion or deletion of the DNA which lies between the two sites

http://www.embo.org/documents/adams.pdf http://jaxmice.jax.org/jaxnotes/archive/501c.html

Or Plasmid Ori Modification of our designed plasmid with a well replicating Ori for eukaryotic cells: http://mcb.asm.org/cgi/content/full/26/3/1124 http://www.pnas.org/content/105/28/9639.full

Inducible promoters Originally derived from bacterial antibiotic response – e.g. tetracycline, streptogramins, coumermycin. http://www.nature.com/mt/journal/v16/n3/full/6300383a.html http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18632760 New systems are based on – rapamycin, Estrogen biotin (19271268 ) Hypoxia http://www.origene.com/cdna/trueorf/pTune-more.aspx ** http://www.bu.edu/abl/files/cell_deans.pdf http://www.neb.com/nebecomm/products/producte3000.asp http://www.clontech.com/products/detail.asp?product_id=157247&tabno=2 Dual regulation (11778900 )

Multiple gene copies Bidirectional promoter (2 flanking genes) Internal ribosome entry site (IRES) from encephalomyocarditis virus, which permits the translation of two open reading frames from a single mRNA transcript. pQuattro (19003423 ) quattrocistronic artificial eukaryotic operons simultaneous and coordinated as well as adjustable expression of up to three independent genes of interest

Subcellular targeting Targeting sequences Nucleus Mitochondria ER cytochrome b5 Golgi Switching FKBP Ariad system ER/Mitochondria http://www.jbc.org/cgi/content/abstract/283/17/11743

Sensing domains ATP sensor Perceval bacterial ATP binding protein, GlnK1 Calcium sensor Oscillations needed Pericam… Hydrogen peroxide sensor HyPer (H2O2) Regulatory domain of the prokaryotic H2O2 sensing protein, OxyRsubmicromolar affinity http://www.evrogen.com/products/HyPer/HyPer.shtml Light White collar -1, phyR, ATM kinase (PMID: 17428747) CFP-YFP ATM phosphorylation site and an FHA phosphospecific binding Specific for ATM over ATR or DNA-PK

Protein life-time Sensor Destabilized FP… http://www.nature.com/nmeth/journal/v6/n3/abs/nmeth0309-188b.html Protein amino-terminal degradation signal

Need to find Peptide based bacterial inducible system Find in literature Build with synthetic biology, test (weeks)

Ideas Inducible KO/KI Cre/FLP induction for response Stem cell Cardiac cell generation in response to oxidative stress! Promoter – MHC  FP Sensor of H2O2  iES cassette  antioxidant response Characterize Calcium sensor ATP sensor

Engineered autophagy receptors Ubiquitin- and LC3-binding protein regulates activation of NFKB by TNF-alpha Responsible for specific autophagy, along with NBR1 target autophagy tag P62- fusion Modified ATG genes Tag-fusion P62-fusion

Inducible transcription system Ariad (drug) http://www.ariad.com/wt/tertiarypage/kits_info