Beta BrainstormingBeta BrainstormingDmitry and ZhizhenDmitry and Zhizhen

1.Colour Bacterial Photography1.Colour Bacterial Photography2.Biofilm Control2.Biofilm Control3.Bacterial weather forecast3.Bacterial weather forecast4.Symbiotic Bacteria in the blood4.Symbiotic Bacteria in the blood5. More wacky ideas5. More wacky ideas

Colour PhotographColour Photograph► Use biobricks from U

T-Austin 2006 project.► Detection of different

wavelength► Bacterial palette

Use Biobricks from UT-Austin 2006Use Biobricks from UT-Austin 2006

► Use biobrick Cph8, (Use biobrick Cph8, (I15010I15010) which is an en) which is an engineered fusion between a cyanobacterial gineered fusion between a cyanobacterial light sensing phytochrome (Cph1) and an light sensing phytochrome (Cph1) and an E.coliE.coli transmembrane histidine kinase, (E transmembrane histidine kinase, (EnvZ). 660nm light causes an isomerization nvZ). 660nm light causes an isomerization in the Cph1 domain of the chimera which iin the Cph1 domain of the chimera which inactivates the histidine kinase acitity of Enactivates the histidine kinase acitity of EnvZnvZ. .

►Edge detectorEdge detector

Detection of different Detection of different wavelengthwavelength

From Yijin’s IdeaFrom Yijin’s Idea► Red light: can be detected by cph8 (Red light: can be detected by cph8 ([2][2], [3]) , [3]) ► Green light: can be detected by bacteriorhodopsGreen light: can be detected by bacteriorhodops

in ("The bacteriorhodopsin molecule is purple ain ("The bacteriorhodopsin molecule is purple and is most efficient at absorbing green light (wavnd is most efficient at absorbing green light (wavelength 500-650 nm, with the absorption maximelength 500-650 nm, with the absorption maximum at 568 nm.", from [4]) um at 568 nm.", from [4])

► Blue light: blue-light photoreceptor phototropin Blue light: blue-light photoreceptor phototropin - nph1 ([5], but this applies for Arabidopsis only. - nph1 ([5], but this applies for Arabidopsis only. Possible to transfect this into E. coli?) Possible to transfect this into E. coli?)

Detection of different Detection of different wavelengthwavelength

► Bacteria can also be Bacteria can also be engineered to engineered to respond to UV light respond to UV light and infrared light.and infrared light.

► Maybe we can Maybe we can reproduce those reproduce those astrological pictures astrological pictures using bacteria.using bacteria.

Bacterial PaletteBacterial Palette

► Red, orange yellow colour-Molecular breeding of carotenoid

► biosynthetic pathways► Blue-lacZ on X-gal► Green/blue – tetrapyrrole► Porphyrin ► Permutations and combi

nations of certain bits of DNA produces different shades of brown

. Cell pellets of E. coli transformants expressing wild-type and mutant cyclases. (A) JM109 carrying plasmid pUC-crtYEU or pUCY2,together with pAC-crtEEU-crtBEU-crtIEU or pAC-crtEEU-crtBEU-I14.(B) JM109 transformants carrying pAC-crtEEU-crtBEU-I14 and variouscyclase mutants.

Biofilm ControlBiofilm Control► Destroy extracellular matrixDestroy extracellular matrix► Enzymes or Molecules can be usedEnzymes or Molecules can be used

Polysaccharide depolymerasePolysaccharide depolymerase► Phage glycanases are very specific. Action of enzyme wasobserved Phage glycanases are very specific. Action of enzyme wasobserved

when added to the phage-susceptible monospecies biofilm, leading when added to the phage-susceptible monospecies biofilm, leading to substantial biofilm degradation (Hughes et al., 1998)to substantial biofilm degradation (Hughes et al., 1998)

A 60 min treatment with a polysaccharase caused a 20% reduction iA 60 min treatment with a polysaccharase caused a 20% reduction in dual-species biofilm adhesion (Skillman et al., 1999)n dual-species biofilm adhesion (Skillman et al., 1999)

NaCl, CaClNaCl, CaCl22, MgCl, MgCl22► Increasing the ionic strength of the medium presumably screens ouIncreasing the ionic strength of the medium presumably screens ou

t cross-linking electrostatic interactions, diminishing biofilm cohesit cross-linking electrostatic interactions, diminishing biofilm cohesiveness (Chen & Stewart, 2002)veness (Chen & Stewart, 2002)

UreaUrea► Treatment with urea caused a 46% reduction in the apparent viscosTreatment with urea caused a 46% reduction in the apparent viscos

ity of the biofilm suspension, suggesting a role for hydrogen bondinity of the biofilm suspension, suggesting a role for hydrogen bonding in cross-linking the biofilm (Chen & Stewart)g in cross-linking the biofilm (Chen & Stewart)

Biofilm ControlBiofilm Control► Biofilm-control strategies based on enzymBiofilm-control strategies based on enzym

ic disruption of the extracellular polymeriic disruption of the extracellular polymeric substance matrix-a modelling study. (By c substance matrix-a modelling study. (By Joao B. Xavier et al)Joao B. Xavier et al)

Biofilm controlBiofilm control► Produce a genetically engineered strain of E.coli Produce a genetically engineered strain of E.coli

which secrete those molecules or enzymes that which secrete those molecules or enzymes that can degrade the extracellular matrix.can degrade the extracellular matrix.

► These bacteria feed on the matrix elements of thThese bacteria feed on the matrix elements of the biofilm. The glycolys pathway needs to be knoe biofilm. The glycolys pathway needs to be knocked out. So when they run out of nutrient the incked out. So when they run out of nutrient the introduced non virulent strain will die.—suicide btroduced non virulent strain will die.—suicide bacteriaacteria

► If the bacteria don’t die and enter a passive staIf the bacteria don’t die and enter a passive state such as endospore or cystae stage, the it will ste such as endospore or cystae stage, the it will stay inactive until the biofilm reappears.tay inactive until the biofilm reappears.

Biofilm ControlBiofilm Control► Preventing cell-cell siPreventing cell-cell si

gnallinggnalling Effect of aiiA expressioEffect of aiiA expressio

n on reducing motility n on reducing motility of P. aeruginosaof P. aeruginosa

aiiA expression does naiiA expression does not reduce adhesion of ot reduce adhesion of P. aeruginosaP. aeruginosa Effect of aiiA expression on

swarming motility of P. aeruginosa PAO1.Ten microlitres of overnight cultures ofPAO1/pME6000 (left) and PAO1/pME6863(right), respectively, were deposited onsemisolid agar plates (see Methods) andincubated at 37 °C for 24 h.

aiiA GeneaiiA Gene Virulence in the opportunistic human pathogen Pseudomonas aeruginosa is controlled by

cell density via diffusible signalling molecules (‘autoinducers’) of the N-acylhomoserine lactone (AHL) type. Two Bacillus sp. isolates (A23 and A24) with AHL-degrading activity were identified among a large collection of rhizosphere bacteria.From isolate A24 a gene was cloned which was similar to the aiiA gene, encoding an AHL lactonase in another Bacillus strain. Expression of the aiiA homologue from isolate A24 in P. aeruginosa PAO1 reduced the amount of the quorum sensing signal N-oxododecanoyl-L-homoserine lactone and completely prevented the accumulation of the second AHL signal, Nbutyryl-L-homoserine lactone. This strongly reduced AHL content correlated with a markedly decreased expression and production of several virulence factors and cytotoxic compounds such as elastase, rhamnolipids, hydrogen cyanide and pyocyanin, and strongly reduced swarming. However, no effect was observed on flagellar swimming or on twitching motility, and aiiA expression did not affect bacterial adhesion to a polyvinylchloride surface. In conclusion, introduction of an AHL degradation gene into P. aeruginosa could block cell–cell communication and exoproduct formation, but failed to interfere with surface colonization.

► --Abstract from Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1

Biofilm controlBiofilm control►Interesting point

enzymic AHL destruction by aiiA expression offers a possibility to examine the role of AHL-dependent quorum sensing without the need for mutant construction. AiiA-dependent signal destruction may therefore be a particularly useful tool to study the impact of quorum sensing in Gram-negative bacteria having multiple AHL regulatory circuits.

Bacterial Weather ForecastBacterial Weather Forecast

► Constructe a half adder to compare TemperaturConstructe a half adder to compare Temperature/humidity states on lawn of bacteriae/humidity states on lawn of bacteria

► Use the biobrick of half adder from ETH-Zurich 2Use the biobrick of half adder from ETH-Zurich 2006006

► Use biobrick J5530 to build a thermometerUse biobrick J5530 to build a thermometer

► Hard to get bacteria to respond to humidity becHard to get bacteria to respond to humidity because they are in culture.ause they are in culture.

Symbiotic Bacteria in the Symbiotic Bacteria in the Blood Blood

► The strain will need to posses the same surface antigen The strain will need to posses the same surface antigen as the host. This means a different strain for each host. as the host. This means a different strain for each host.

► The strain probably should not possess any the ability to The strain probably should not possess any the ability to metabolize glucose, glycogen or fatty acids (or do it very metabolize glucose, glycogen or fatty acids (or do it very slowly). It should be able to metabolize a number of coslowly). It should be able to metabolize a number of common toxins such as alcohol. mmon toxins such as alcohol.

► The polymerase enzymes of the symbiote will need to bThe polymerase enzymes of the symbiote will need to be thermophilic (activated at temperatures above normal e thermophilic (activated at temperatures above normal body temperature). This will prevent the strain from divibody temperature). This will prevent the strain from dividing uncontrollably. ding uncontrollably.

► This could be expanded to cover a number of toxins, proThis could be expanded to cover a number of toxins, providing that the first tree points are obeyed. viding that the first tree points are obeyed.

More wacky ideasMore wacky ideas

► 1. Use Citrobacter to collect heavy metal s1. Use Citrobacter to collect heavy metal salts out of solution, such as uranium whicalts out of solution, such as uranium which is converted to uranyl phosphate (originh is converted to uranyl phosphate (original research by University of Birmingham). al research by University of Birmingham).

► 2. Another idea was to use bacteria to det2. Another idea was to use bacteria to detect chemicals and release YFP when they ect chemicals and release YFP when they do.do.