Team:Groningen/Expression

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Expression of chaplins

Summary

The goal of our project is to let Bacillus subtilis make a hydrophobic coating by forming a biofilm and then expressing and secreting chaplins. However, first we needed to test whether B. subtilis was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in B. subtilis using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want B. subtilis to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in B. subtilis; one that gets triggered in late exponential growth (srfA operon) and one that is involved in the formation of biofilm (yqxM-sipW-tasA operon). Using the srfA promoter (BBa_K305007), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.


Subtilin induced expression of chaplins

The biofilm forming capacity of Bacillus subtilis makes it a good host for our application. In addition, B. subtilis is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both B. subtilis and Streptomyces coelicolor are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in B. subtilis. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression. We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for B. subtilis and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in B. subtilis, and flanked these constructs with the biobrick prefix and suffix.

SURE expression system

Subtilin induction of GFP by the SURE system (Bongers et al, 2005)

Because it is uncertain how chaplin expression will affect B. subtilis, the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers et al, 2005). This system uses the subtilin sensing machinery present in a strain of B. subtilis that autoinduces the production of more of the lantibiotic subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called spa boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem et al, 2004). In the SURE system, a B. subtilis strain naturally lacking the subtilin biosynthesis genes has the spaRK genes introduced into its genome. A plasmid carrying a spa box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling.

Subtilin induced GFP expression in Bacillus subtilis using the BBa_K305011 backbone

We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated spaS promoter, producing our subtilin inducible expression backbone part, BBa_K305011. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part BBa_E0240 into the BioBrick site and induced liquid cultures of B. subtilis carrying this plasmid (and the spaRK genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of B. subtilis. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins


Chaplin detection
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and TCA precipitated supernatant with TFA to purify chaplin proteins. TFA treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.


Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called Thioflavin T. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.

Using this method we succesfully detected chaplins C, E and H in purified cell walls from induced B. subtilis cultures, confirmation of these results was provided by malditov mass spectrometry.

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Purified chaplins from S. coelicolor exitate at 482 when stained with THT. Different dilutions were used to set up a ladder.
Purified E and H chaplins from B subtilis exitate at 482 when stained with THT
Purified C chaplins from B subtilis exitate at 482 when stained with THT
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Purified E chaplins from B subtilis exitate at 482 when stained with THT

[[Image:MassSpec.jpg|700px|thumb|none|Mass spectometry results confirm the production of Chaplin H (5128) and E (5144) in Bacillus (green). Peaks is Streptomyces (blue) corrispond, although the peak of chaplin E (5144) indicates that somewhere during translation or in post-translational processes a 21kb threonine group is lost. The non induced B. subtilis strain serves as a control (red). Chaplin C has not been detected this way, but that was not to be expected (Claessen et al. (2009).]] ===Timed expression of chaplins in a biofilm === An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. [[Image:Groningen-Promotors-sketch.png‎|300px|left]] [[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]] '''''srfA''''' The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [http://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. [[Image:florescence_srfA.jpg]] '''''yqxM''''' [[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]] The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work. ===References=== Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878] Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers. 2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424] Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17] Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261] Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766] Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]