1. 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

2. 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

3. RNAi
Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch

4. Stable integration sites (Multiple)
Integration site-specific transcriptional reporter gene analysis using Flp recombinase targeted cell lines.
Biotechniques Feb;42(2):
Stable acceptor cell line(s)
GOI/sensor/shRNA #1
GOI/sensor/shRNA #2
Bidirectional/multiple CDS…. (high complexity)

5. 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

7. Or Plasmid Ori
Modification of our designed plasmid with a well replicating Ori for eukaryotic cells:

8. Inducible promoters
Originally derived from bacterial antibiotic response – e.g. tetracycline, streptogramins, coumermycin.
New systems are based on –
rapamycin,
Estrogen
biotin ( )
Hypoxia **
Dual regulation ( )

9. 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 ( )
quattrocistronic artificial eukaryotic operons
simultaneous and coordinated as well as adjustable expression of up to three independent genes of interest

10. Subcellular targeting
Targeting sequences
Nucleus
Mitochondria ER
cytochrome b5
Golgi
Switching
FKBP Ariad system
ER/Mitochondria

11. 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
Light
White collar -1, phyR,
ATM kinase (PMID: )
CFP-YFP
ATM phosphorylation site and an FHA phosphospecific binding
Specific for ATM over ATR or DNA-PK

12. Protein life-time
Sensor
Destabilized FP…
Protein
amino-terminal degradation signal

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

14. 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

15. 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

16. Inducible transcription system
Ariad (drug)