A Biofilm Coating By

Hydrophoboﬁlm!

A bioﬁlm coating by Chemical coatings can be harmful Heavy metals accumulate in ecosystems Bioﬁlm coatings are a good alternative • Green • Cheap • Easy Hydrophobic properties prevent fouling Surface hydrophobicity in Streptomyces coelicolor Chaplins are strongly hydrophobic proteins

Monomers assemble into extremely persistent amyloid ﬁbers

Chaplins a group of hydrophobic proteins

Chp A-C Large(±225 AA)

Hydrophobic Hydrophillic cellwall anchor Chp D-H Small(±63 AA)

Hydrophobic

* Claessen D, Rink R, de Jong W, Siebring J, de Vreugd P, Boersma FG, Dijkhuizen L, Wosten HA, Genes and Development 2003 17 (14): 1714-1726. Chaplins: Biobricks

Chp E & H

Chp C

Chp *C

Sortase recognition site & Sortase from S. aureus B. subtilis is a suitable host

WT Rok degU • Forms relatively rigid bioﬁlms • Fast growing • Gram positive • Model organism • Expertise Chaplin expression via spaRK Reliable expression system for B. subtilis

GFP expression by spaRK

120000

100000

80000 intensity

units) 60000 GFP1 40000 GFP2 arbitrary ( 20000 Fluorescence 0 0 0,5 1 2 4 8 Subtilin percentage (v/v) Chaplin detection Thioﬂavin T: Amyloid speciﬁc staining

Puriﬁcation of chaplins by total cell disruption and boiling in SDS.

ThT binds assembled chaplins, with ﬂuorescent emissions between 460-600 nm, with an emission peak around 482 nm

Chaplin detection with ThT

Chp-E detected emission

Chp-C detected emission Mass spectrometry conﬁrmed successful expression of mature chaplins Bioﬁlm formation

1. Biomass accumulation Bioﬁlm formation

1. Biomass 2. Adhesion accumulation Bioﬁlm formation

1. Biomass 2. Adhesion 3. ECM accumulation formation Bioﬁlm coating

1. Biomass 2. Adhesion 3. ECM accumulation formation Quorum sensing ComX signaling ComX signaling comX expression model Density dependent srfA promotor activity

pAD 1 parstability function 653 of the Sok±antisense R N A (Thisted et al., 1 9 9 5 ; Severallines ofevidence supportthe hypothesisthat Franch and G erdes, 1996). As the hok± sok duplex is par is a P S K s y s te m w ith R N A I e n c o d in g th e to x in a n d rapidly cleaved by RNase III (G erdes et al., 1 9 9 2 ), th is RNA IItheantidote(Weaver,1995;W eaveretal., 1 9 9 6 ; is necessary to ensure that a poolof hok messageis 1998).U ntilnow , w e have been uncertain w hetherthe available to be activated in plasm id-free cells. The hok Fst peptide encoded by RNA I is translated and is messageisslowlydegradedfrom the30 end by host re s p o n s ib le fo r par-in d u c e d k illin g . In th is re p o rt, w e RNases,eventuallyresulting inremovalofthe fb i.Once provide evidence show ing that this sm allpeptide is th e fb i re g io n h a s b e e n p ro c e s s e d , hok can either be in d e e d th e par to x in . In a d d itio n , w e s h o w th a t R N A II is bound by the sok antisense R N A or, in plasm id-free capable of preventing the translation of fs t by inhibiting cells,can be translated to produce toxin. A m etastable ribosom e binding.Finally,w e dem onstrate thatR N A IIis hairpin has also been show n to form at the 50 end of less stable than RNA I,as would be expected for the th e n a s c e n t hok tra n s c rip t. T h is s tru c tu re s e q u e s te rs antidote ofa PSK system. th e mok tra n s la tio n in itia tio n re g io n u n til tra n s c rip tio n of hok is c o m p le te d a n d th e fb i re g io n c a n s u p p re s s tra n s la tio n (N a g e l et al., 1 9 9 9 ). Results Par is a 4 0 0 b p d e te rm in a n t re q u ire d fo r th e s ta b le The parto x in is e n c o d e d b y fs t in h e rita n c e o f th e Enterococcus faecalis plasm id pAD 1 (W e a v e r et al., 1 9 9 3 ). T h e par determinanthas been The RNA I gene has been shown to be the toxic show n to stabilize plasm ids in the host population by a com ponent of the par system but,as discussed above, PSK m echanism (Weaver,1995;W eaver et al., 1 9 9 6 ; whethertheRNA I-encodedpeptide(Fst)orsome other 1998)and isthefirstplasm idstabilitysystem ofthiskind attribute ofR N A Iisresponsibleforthistoxicity has not id e n tifie d in a G ra m -p o s itiv e o rg a n is m , w ith th e e x c e p tio n been determined.To resolve thisquestion,w e soughtto of restriction-modification systems (Naito et al., 1 9 9 5 ). determine w hether fs t was sufficientforcellkilling.As The par lo c u s e n c o d e s tw o s m a ll R N A tra n s c rip ts , show n in Table 1, pD A K 704, a pA M 401 construct designated R N A Iand R N A II,w hich are essentialforits containing the com plete R N A I gene, could not be fu n c tio n (W e a v e r a n d T ritle , 1 9 9 4 ). R N A I a n d R N A II a re in tro d u c e d in E.faecalis UV202cellsunlessRNA IIwas convergently transcribed from prom oters located ateach already present on another plasm id (pDAK611). To end of par,andterminateata commonbidirectional determinew hetherthe Fstpeptidew as sufficientforcell te rm in a to r (F ig . 1 ). T h e R N A s a re tra n s c rib e d in o p p o s ite killing, the 3 0 non-coding sequence of R N A I w as directions across a pair of direct repeat sequences re m o v e d , s o th a t o n ly th e p ro m o te r, S h in e ± D a lg a rn o lo c a te d a t th e 5 0 end of each gene. This50 re g io n a n d (S D ) s e q u e n c e a n d fs t open reading frame remained. the shared term inator region provide regions ofcom ple- Thisshortened RNA I gene was cloned into pAM 401 mentarityatwhichthetwoRNAscouldinteract.Computer- creating pD A K 614. Transform ation experiments w ith this predictedR N A secondarystructureanalyses indicatethat clone gave results similar to those for the w ild-type these regions are presenton exposed loops in both RNAs RNA Iclone (Table1),indicating thatitwas stilllethal makingthem accessibleforinteraction(Fig.2).Addition- to E. faecalis and thatR N A IIw as stillprotective. In ally, R N A I codes forthe peptide Fst (fa e c a lis plasm id- contrast,a construct in w hich the finalcodon of fs t was stabilizing to x in ). T h e tra n s la tio n in itia tio n s ig n a ls fo r fs t replaced by a stop codon (pDAK615) could be areadjacenttothe50 com plem entary repeats,suggesting in tro d u c e d in to E. faecalis cells in the absence of th a t its tra n s la tio n w o u ld b e in h ib ite d b y in te ra c tio n w ith RNA II.The transformation efficiencyofpDAK615 was RNA II.Thus,theorganizationofthe parlo c u s s u g g e s ts consistently,but not significantly,low er than that of the th a t R N A II re g u la te s th e tra n s la tio n o f th e R N A I-e n c o d e d pAM 401 vector control in the absence of R N A II fs t via an antisense R N A m echanism.Killswitch modeling( P . 0.05), but transformant colonies w ere m uch

Fig.1.Features ofthe pAD1 parstability determ inant.Prom oters forR N A Iand R N A IIare indicated by black arrow s on each end.The open re a d in g fra m e , fs t, encodes• fst fora encodes 33-amino-acidpeptide.The a two R N As read inoppositedirections across directrepeatslabelledD R a and DRb (arrows)toa bidirectionalterminator(convergingarrows).Thetoxic compound SpeIsiteused tocreateRNA Ideletion m utantsism arked. Q 2000 Blackw ellScience• RNAII Ltd,MolecularMicrobiology binds RNAI, 37 ,652±660 • Diference in half life time Hydrophoboﬁlm

Bioﬁlm Chaplin expression Kill switch formation Applications Integration of iGEM projects

iGEM team Groningen 2010

Applying a bioﬁlm on a surface by making a viscous medium paste Integration of iGEM projects

example: iGEM team iGEM team Newcastle Groningen 2010 2010

Applying a biofilm Concrete repairing on a surface by B. subtilis making a viscous medium paste Biofilm coating: versatile machine Biofilm coating: versatile machine Human practices

• Ethics & Safety

• Awareness: – Presentations at schools – Hands on lab experience – Debate (knowledge cafe) Acknowledgements Our Instructors / Advisors • prof. dr. Oscar Kuipers: Molecular Genetics (Head) • dr. Jan Willem Veening: Molecular Genetics • dr. Dennis Claessen: Microbial Physiology • prof. dr. Roel Bovenberg: Synthetic biology and Cell engineering • Martijn Herber: Molecular Genetics • Auke van Heel: Molecular Genetics

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