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

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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

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SLIDE 1
  • C. imager
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SLIDE 2
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SLIDE 3

C.ima mager

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SLIDE 4

C.ima mager

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SLIDE 5

C.ima mager

Colo lor mim imic Motion control Caulobacter crescentus

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SLIDE 6

Colo lor mim imic

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SLIDE 7

C.ima mager

Colo lor mim imic Motion control Caulobacter crescentus

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SLIDE 8

Motion control

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SLIDE 9

C.ima mager

Colo lor mim imic Motion control Caulobacter crescentus

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SLIDE 10

YF YF1 FixJ ixJ Fix ixK2 Pλ BF BFP Pcp cpcG cG2 GFP λ CI

RFP

Pλ

Lig ight-inducible cir ircuits

GREEN RED BLUE

Evan J Olson，Nature Methods 11,2014

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SLIDE 11

Florescent proteins produced directly without sensing lights

Results----

  • --- pigment
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SLIDE 12

Results----

  • --- Blue circuits
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SLIDE 13

Results----

  • --- Green Circuits
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SLIDE 14

Results----

  • --- projecting imaging and presenting image
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SLIDE 15

Problem: Bacteria may not present the image we project!

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SLIDE 16

Desired Image RGB Components Protein

  • utput

Light Input

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SLIDE 17

Desired Image RGB Components Protein

  • utput

Light Input Protein

  • utput
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SLIDE 18

P-protein output I-light intensity

Evan J Olson，Nature Methods 11， 2014

11

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

  • 10

10 20 30 40 50 60 70 80 90

80 20

Light intensity(w/m^2) Protein output

Desired Image RGB Components Protein

  • utput

Light Input

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SLIDE 19

R G B

Desired Image RGB Components Protein

  • utput

Light Input

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SLIDE 20

Desired Image RGB Components Protein

  • utput

Light Input

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SLIDE 21

C.ima mager

Colo lor mim imic Motion control Caulobacter crescentus

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SLIDE 22

Caulobacter Crescentus

An amazing vector for bacterial photography

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SLIDE 23

Adhesive ability of Caulobacter crescentus

hold

  • ldfast

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

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SLIDE 24

Stalked Cell: Holdfast Swarmer Cell: flagellum

Regulation: More stalked cell

Special lifecycle

Pamela J B Brown, . Advances in microbial physiology. 2009

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SLIDE 25

Validation of adhesive ability of C.crescentus

Gram Staining

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SLIDE 26

Validation of adhesive ability of C.crescentus

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

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SLIDE 27

Motion Control - Flagellum Rotation

DgrA/ DgrB

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SLIDE 28

Motion Control - Holdfast Biosynthesis

Hfi fiA

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SLIDE 29

Design of Blue C.imager

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SLIDE 30

C.Imager simulation

  • E. coli, 48h
  • C. crescentus , 48h
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SLIDE 31

Conjugation

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SLIDE 32

Conjugation

  • E. coli S17-1(Wit

ith Plasmids)

  • C. C. (Wit

ith Plasmids)

  • C. C.

F Plasmid (With OriT) Color Motion

？

In Init itial l Concentration

? ?

Resulting Bacteria

  • Fang

Teng; Barbara E Murray; George M Weinstock, Plasmid. 1998 . 10.1006/plas.1998.1336

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SLIDE 33

ODE Model

𝑒𝑜1 𝑒𝑢 = 𝑠 1 1 − 𝑜1+𝑜2+𝑜3 𝐿

− 𝑒1 𝑜1

𝑒𝑜2 𝑒𝑢 = 𝑠 2 1 − 𝑜1+𝑜2+𝑜3 𝐿

− 𝑒2 − 𝑒3 𝑜2 − 𝑙𝑜1𝑜2

𝑒𝑜3 𝑒𝑢 = 𝑠 2 1 − 𝑜1+𝑜2+𝑜3 𝐿

− 𝑒2 𝑜3 + 𝑙𝑜1𝑜2

𝐶𝑗𝑠𝑢ℎ 𝐸𝑓𝑏𝑢ℎ 𝐷𝑝𝑛𝑞𝑓𝑢𝑗𝑢𝑗𝑝𝑜 𝐷𝑝𝑜𝑘𝑣𝑕𝑏𝑢𝑗𝑝𝑜 Concentration

Change

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SLIDE 34

Parameters

Parameters From Experiments

𝒔𝟐 𝒔𝟑 𝒆𝟐 𝒆𝟑 𝒆𝟒 0.30 0.40 0.06 0.06 0.02 𝒍 𝑳 0.5 1.5

𝒔 − 𝒄𝒋𝒔𝒖𝒊 𝒆 − 𝒆𝒇𝒃𝒖𝒊 𝑳 − 𝒅𝒃𝒒𝒃𝒅𝒋𝒖𝒛 𝒍 − 𝒅𝒑𝒐𝒌𝒗𝒉𝒃𝒖𝒋𝒑𝒐

𝑢/ℎ 𝑜 30 15 1.2 0.6

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SLIDE 35

Results

1.2 0.6 𝑜 𝑢/ℎ 100 60 24

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SLIDE 36

0.0 0.5 1.0 n1 0.0 0.5 1.0 n2 0.0 0.5 1.0 n3

𝐹𝑜𝑒 𝑇𝑢𝑏𝑠𝑢

Results in Phase Space

𝑜3 𝑜1 𝑜2

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SLIDE 37

Phase-space Analysis

1.

  • 1. Whatever the initial concentration,

C.c .c. (W (Wit ith plas lasmid ids) will domin inate the system finally. 𝑜3 𝑜1 𝑜2

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SLIDE 38

𝑜1 𝑜2

Phase-space Analysis

  • 2. Putting more C.c

.c. th than E. . coli li initially will acc accelerate the dominance. 𝑜3 𝑜1 𝑜2

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SLIDE 39

Results of Conjugation

RedC.imager BlueC.imager

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SLIDE 40

RNA Logic Gates

To make parts work faster and more accurate

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SLIDE 41

Advantages

Fast Efficient Less Cross Talk

Hammerhead Ribozyme

RNA Regulation

Robert Penchovsky Computational design and biosensor applications

  • f

small molecule-sensing allosteric

  • ribozymes. Biomacromolecules. 2013 . 10.1021/bm400299a
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SLIDE 42

Safety

Kill Switch

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SLIDE 43

Safety Design: Kill Switch

LPS Nutrition LALF

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IPTG LALF(Lac I Anti-LPS Factor)

IPTG Lac 1 LALF

TAT Signal Peptide

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SLIDE 45

Result

Growth of E. coli in gradient IPTG medium

Amount of E. coli decreases Concentration of IPTG increases

0.1% IPTG 0

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SLIDE 46

Summary

The work we have done

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SLIDE 47

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).

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SLIDE 48

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.

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SLIDE 49

Parts Construction

Blue and red light sensing-imaging system

BB BBa_K1363400 BBa_K1363401

Motion control

BB BBa_K1363000 BBa_K1363001 BB BBa_K1363002 BBa_K1363005

Kill switch based on LALF regulation

BB BBa_K1363200 BBa_K1363201 BB BBa_K1363003 BBa_K1363004 BBa_K1363006

Conjugation parts from E.coliS17-1 to other bacteria RN RNA Lo Logic Ga Gates

BB BBa_K1363610 BBa_K1363611 BBa_K1363606 BBa_K1363607 BBa_K1363609 BBa_K1363610 BBa_K1363602 BB BBa_K1363603 BBa_K1363604 BBa_K1363605 BBa_K1363601 BBa_K1363600 BBa_K1363500

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SLIDE 50

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.

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Policy & Practice

C.imager, not only in lab, but in public.

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Making ambers & Rubbing plants

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SLIDE 53

Newsletter

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Outreach

Nanjing Taiwan Peking China

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Lab open day

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SLIDE 56

Team

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SLIDE 57

Advisors Jiong Hong Haiyan Liu Zhi Liang

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SLIDE 58

Sponsors

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SLIDE 59

C.imager, view colorfully

Thanks for Listening 

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SLIDE 60

Q&A Section

Sponsors