A genetically encoded system for programmable cell assembly using surface-displayed DNA oligonucleotides
Yuhao Min1, Tong Si2, Huimin Zhao1,2
1. Department of Chemistry2. Department of Chemical and Biomolecular Engineering
University of Illinois at Urbana-Champaign
[1] Z Gartner, et al, PNAS, 2009, 106(12), pp4606-4610[2] www.nei.nih.gov[3] F Akter, et al, Anal Chem, 2012, 84 (11), pp5040–5046[4] F Farzadfard, et al, Science, 2014, 346(6211), pp1256272-3; http://utminers.utep.edu/rwebb/html/rolling_circle_replication.html; X Tan, et al, Biochemistry, 2004, 43 (4), pp 1111–1117[5] https://www.semrock.com/flow-cytometry.aspx.
3. Results
Conclusion
Project Goal: to develop a method that enables cell todisplay DNA and facilitates their assembly.
1. Introduction
• Phage protein φX174 A* isfused with a membraneprotein as the ssDNA anchor.
• ssDNA molecules generatedin vivo can be covalentlybounded to a phage proteinφX174 A* .
Fig. 1.2 Anchored phage gene A* attaches ssDNA [3]
Fig.1.3 Schemes of ssDNA generation[4]
• Three systems were employed to generated ssDNA in vivo.1. Multicopy single-stranded DNA (msDNA)2. Moloney leukemia viral reverse transcriptase (MoMuLV RT)3. M13 bacteriophage reverse transcriptase (M13 RT)
2.1 Strains construction
• Phage protein φ147A* gene was fused with E.coli outer membrane lipoprotein gene Blc.• Three reverse transcriptase (RT) genes were cloned separately.• Customized oligonucleotide sequences were introduced in the plasmids.• Protein expression was induced by IPTG under the control of the lac operon.• The plasmids were confirmed by diagnostic digestion and DNA sequencing.
Fig. 2.1 DNA construct for displaying ssDNA on cell surfaces strains
2.2. ssDNA detection• Flow cytometry was used to examine
transformed cells.• ssDNA was stained using a membrane
impermeable dye (propidium iodide, PI),so that only cell surface-displayed DNAcan be stained.
• Treatment with exonucleas I (ExoI) willdigest ssDNA at cell surfaces and reducefluorescence.
• Sequence-complementary ssDNA wasexpressed in two cells and stained withdifferent fluorophores. (DAPI and FITC)Mixing two kinds of cells promotes cellbinding that can be detected with flowcytometry.
• Designed plasmids were successfully constructed and expressed in E.coli cells• Cells were enabled to display custom ssDNA sequences onto cell surfaces
without complex modification or chemical functionalization• The existence of surface-bound ssDNAs and cell self assembly were confirmed
using flow cytometry, confirming a functional genetic system was established for in vivo displaying of ssDNA molecules on cell surfaces
• Precise patterning of cells in three dimensions is highly desirable inboth basic and applied research for tissue engineering.
• Current bottom-up methods requires complicated chemicalfunctionalization protocols to attach oligonucleotides.
• A genetic encoded system is devised to covalently attachcustomizable single-stranded DNA(ssDNA) onto Escherichia coli cellsurface.
Fig. 1.1 Cell-self display ssDNA of interest [1]
DAPI95.7
FITC96.0
DAPI95.7
C98.2
FITC95.7
C15.4
• DNA sequencing results of plasmids revealed expected nucleotide sequences ofinserted fragments.
• Digestion with ExoI reduced fluorescence intensities for all systems,confirming the existence of ssDNAs on cell surfaces.
• Complementary ssDNA molecules were expressed on different cells to allowhybridization and cell assembly, revealed by dual fluorescence straining.
Fig. 3.2 Flow cytometry profile of differently stained cell surface ssDNA
Fig. 3.3 Fluorescence intensity of cells when treated with ExoI
Fig. 2.2 Scheme of flow cytometer [5]
Fig. 3.1 Sequencing data of M13,Ec86 and MoMuLV plasmids
−Thank Dr. Tong Si, Prof. Huimin Zhao, and Prof. Yi Lu.−Y.M. thank John E. Gieseking Scholarship from the Department of Chemistry.−This project is financially supported by Institute for Genomic Biology.
Acknowledgment
IPTG
Sample nameM13M13 ExoIMuMuLVMoMuLV ExoIEc86Ec86 ExoIControlControl ExoI
Fig. 1.2 Tissues are made up of different cells in spatial arrangement
[2]
References
