C. imager C.ima mager C.ima mager C.ima mager Colo lor mim - PowerPoint PPT Presentation

C. imager

C.ima mager

C.ima mager

C.ima mager Colo lor mim imic Motion control Caulobacter crescentus

Colo lor mim imic

C.ima mager Colo lor mim imic Motion control Caulobacter crescentus

Motion control

C.ima mager Colo lor mim imic Motion control Caulobacter crescentus

Lig ight-inducible cir ircuits GREEN GFP Pcp cpcG cG2 RED λ CI RFP P λ BLUE BF BFP YF YF1 FixJ ixJ P λ Fix ixK2 Evan J Olson ， Nature Methods 11,2014

Results---- ---- pigment Florescent proteins produced directly without sensing lights

Results---- ---- Blue circuits

Results---- ---- Green Circuits

Results---- ---- projecting imaging and presenting image

Problem: Bacteria may not present the image we project!

Desired Protein RGB Light Input Image output Components

Desired Protein Protein RGB Light Input Image output output Components

11 Desired Protein RGB Light Input Image output Components Protein output 90 80 80 70 60 50 40 30 20 20 10 0 P-protein output -10 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 I-light intensity Light intensity(w/m^2) Evan J Olson ， Nature Methods 11 ， 2014

Desired Protein RGB Light Input Image output Components G R B

Desired Protein RGB Light Input Image output Components

C.ima mager Colo lor mim imic Motion control Caulobacter crescentus

Caulobacter Crescentus An amazing vector for bacterial photography

Adhesive ability of Caulobacter crescentus fla flagell llum stalk hold oldfast Strength/ 𝑶 ∙ 𝒏𝒏 −𝟐 Na Name Sh Shear Str SBS Glue 0.8 Rubber with Metal 1.028 AB Glue 8.5 Holdfast of C. crescentus 68

Regulation: More stalked cell Special lifecycle Stalked Cell: Holdfast Swarmer Cell: flagellum Pamela J B Brown, . Advances in microbial physiology. 2009

Validation of adhesive ability of C.crescentus Gram Staining

E. . coli li, 1 1 d growth, 2 2 min in C. . cr crescentus, 1 1 d growth, 2 2 water flo flow, 40 400X 0X min in water flo flow, 40 400X Validation of adhesive ability of C.crescentus

Motion Control - Flagellum Rotation DgrA/ DgrB

Motion Control - Holdfast Biosynthesis Hfi fiA

Design of Blue C.imager

C.Imager simulation E. coli, 48h C. crescentus , 48h

Conjugation

Conjugation Color Init In itial l Concentration ？ F Plasmid C. C. E. coli S17-1(Wit ith Plasmids) ? ? (With OriT) Motion Resulting Bacteria • Fang Teng; Barbara E Murray; George M Weinstock, Plasmid. 1998 . 10.1006/plas.1998.1336 C. C. (Wit ith Plasmids)

ODE Model Concentration 𝐶𝑗𝑠𝑢ℎ 𝐷𝑝𝑛𝑞𝑓𝑢𝑗𝑢𝑗𝑝𝑜 𝐸𝑓𝑏𝑢ℎ 𝑒𝑜 1 𝑜 1 +𝑜 2 +𝑜 3 𝑒𝑢 = 𝑠 1 1 − − 𝑒 1 𝑜 1 𝐿 𝐷𝑝𝑜𝑘𝑣𝑕𝑏𝑢𝑗𝑝𝑜 Change 𝑒𝑜 2 𝑜 1 +𝑜 2 +𝑜 3 𝑒𝑢 = 𝑠 2 1 − − 𝑒 2 − 𝑒 3 𝑜 2 − 𝑙𝑜 1 𝑜 2 𝐿 𝑒𝑜 3 𝑜 1 +𝑜 2 +𝑜 3 𝑒𝑢 = 𝑠 2 1 − − 𝑒 2 𝑜 3 + 𝑙𝑜 1 𝑜 2 𝐿

Parameters From Experiments Parameters 𝒔 − 𝒄𝒋𝒔𝒖𝒊 𝒆 − 𝒆𝒇𝒃𝒖𝒊 𝑳 − 𝒅𝒃𝒒𝒃𝒅𝒋𝒖𝒛 𝑜 𝒍 − 𝒅𝒑𝒐𝒌𝒗𝒉𝒃𝒖𝒋𝒑𝒐 1.2 𝒔 𝟐 𝒔 𝟑 𝒆 𝟐 𝒆 𝟑 𝒆 𝟒 0.30 0.40 0.06 0.06 0.02 𝒍 𝑳 0.6 0.5 1.5 𝑢/ℎ 15 30

Results 𝑜 1.2 0.6 𝑢/ℎ 24 60 100

Results in Phase Space 1.0 n2 𝑜 3 0.5 0.0 𝐹𝑜𝑒 𝑜 2 1.0 n3 0.5 𝑇𝑢𝑏𝑠𝑢 0.0 0.0 0.5 n1 1.0 𝑜 1

1. 1. Whatever the initial concentration, Phase-space C.c .c. (W (Wit ith plas lasmid ids) will domin inate Analysis the system finally. 𝑜 3 𝑜 2 𝑜 1

2. Putting more C.c .c. th than E. . coli li Phase-space initially will acc accelerate the Analysis dominance. 𝑜 3 𝑜 2 𝑜 2 𝑜 1 𝑜 1

Results of Conjugation Red C.imager Blue C.imager

RNA Logic Gates To make parts work faster and more accurate

Hammerhead Ribozyme RNA Regulation Advantages Fast Efficient Less Cross Talk Robert Penchovsky Computational design and biosensor applications of small molecule-sensing allosteric ribozymes. Biomacromolecules. 2013 . 10.1021/bm400299a

Safety Kill Switch

Safety Design: Kill Switch LPS LALF Nutrition

IPTG LALF(Lac I Anti-LPS Factor) IPTG Lac 1 LALF TAT Signal Peptide

Result Amount of E. coli decreases Concentration of IPTG increases 0.1% IPTG 0 Growth of E. coli in gradient IPTG medium

Summary The work we have done

Summary Color Mim imic • Constructed RGB light inducible circuits. • Tested the photographic system with projector. • Calibrated chromatic aberration by modeling. Motion Control • Validated the adhesiveness of C.crescentus. • Extracted motion control parts from chromosomal DNA: DgrA/DgrB and HfiA. • Constructed standard parts for motion control (Flagellum Control & holdfast biosynthesis).

Summary Conjugation • Found optimal initial concentrations in conjugation by modeling. • Developed the protocol of conjugation between C. crescentus and S17-1 E.coli. • Constructed parts for conjugation. • Validated the effectiveness of adhesiveness in raising the resolution of images by modeling. Safety • Constructed kill-switch parts and validated its functionality.

Parts Construction Blue and red light sensing-imaging system RNA Lo RN Logic Ga Gates BB BBa_K1363400 BBa_K1363603 BB BBa_K1363401 BBa_K1363604 Motion control BBa_K1363605 BB BBa_K1363000 BB BBa_K1363002 BBa_K1363601 BBa_K1363005 BBa_K1363001 BBa_K1363600 Conjugation parts from E.coli S17-1 to other bacteria BBa_K1363500 BB BBa_K1363003 BB BBa_K1363610 BBa_K1363004 BBa_K1363611 BBa_K1363006 BBa_K1363606 Kill switch based on LALF regulation BBa_K1363607 BBa_K1363609 BBa_K1363200 BB BBa_K1363610 BBa_K1363201 BBa_K1363602

Future Work Accomplishment of C.imager • Light-induced color mixture tests • Light-induced chromatic pattern tests RNA logic circuit improvement • Introducing RNA logic gates in the imaging system to improve its accuracy and efficiency.

Policy & Practice C.imager, not only in lab, but in public.

Making ambers & Rubbing plants

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