IGEM:IMPERIAL/2008/Bioprinter/Subteam 1

B. subtilis and Motility: Subteam 1

OK. Lots of wetlabs hopefully, but also plenty of trawling for papers... =P Any chance of a bit of modelling maybe???

All Hail the DBTBS[1], the massive database and reference point for the transcription regulation of B.subtilis. All promoters were found using the DBTBS unless otherwise stated.

Many thanks to all involved at the Human Genome Center at Institue of Medical Science at the University of Tokyo. Your database has been invaluable to helping us find much needed information and sequences for parts for our chassis.

To Do

Cross these off when you've done them (or comment next to them) but don't delete them!

Contact

• Newcastle - In progress...Still ongoing
• Hawaii - Now in E.coli, may ask why later...
• Cambridge - Are they really using it much?
• Angelika - In progress....Will discuss tomorrow

Dry Work

• Obtain strains...Just need to go pick them up
• Find Promoters....Done (4), more easily available
• Find/Check ribosome binding sites (RBS)
• Find/Check Terminators...Ongoing
• Protocols: Cambridge 07; Isolation of DNA from B. subtilis...All done!
  • Culturing
  • Characterisation
  • 2nd transformation
  • Heterologous expression
• Find information on vectors
• Strain specifics
  • 168 ..... General workhorse labstrain - We'll be using this. Easily transformable.
  • PY79..... Less useful
• Information on integration loci ..... Come on James!

Wet Work

• Make those buggers work correctly
• etc.

Hardware

Strains

At Imperial we have access to two strains, 168 and PY79.

B.subtilis 168 is a common lab strain of B. subtilis[2] and should be well suited for growth and our use, we will need to characterise in in order to model clutch activity and swimming ability.

It may also be possible to obtain a strain from the US which is already ΔespE and would so save our team the time and effort that would be involved in knocking out the epsE gene. However the strain of this B. subtilis is NCIB 3610[3] (see supplementary material of reference), which is a wild strain and so will not be adapted for the lab thus being harder to culture and transform.

If the wild strain knockout is unusable we will be required to manually knock out the epsE gene or find some way to inhibit its transcription or translation.

Vectors

E. coli plasmids cannot replicate in B. subtilis and so regular biobricks are not usable in such a chassis.

In labs, constructs called shuttle primers capable of replicating in both species are used and often created by merging together an E.coli vector and a B.subtilis vector. However a potentially easier and more useful method is to use a vector that will integrate itself into the B.subtilis chromosome , bypassing the need to have multiple vectors in a single cell and the risk that B.subtilis will digest the vector as it sometimes does with plasmid vectors produced in E.coli.

Integration Loci

The strains we will get access to from Imperial are B. subtilis 168 and PY79. The vectors we will pick up are for integration at locus amyE, a commonly used locus in B. subtilis. To complement this we may use locus lacA which is close to the amyE locus but does not interfere with it[4]. The details regarding integration (ie. insert size, flanking sequence etc.) will be looked up tomorrow...

Software

Circuitry Issues

It is worth noting that studies on B.subtilis expression have discovered that in a polycistron, although each gene has its own Ribosome Binding Site, each gene down the polycistron will be less highly transcribed[5].

This may cause problems if we decide to put multiple genes after a single promoter

Promoters

The publishing of the B. subtilis genome[6] may allow simple ways to obtain potentially useful sequences for the B. subtilis chassis

And on the 8th day, the spirits gave us the University of Tokyo and they compiled the DBTBS[1] and all was well...

Seriously, the DBTBS is a huge but very user-friendly database of all things gene regulation in B.subtilis, many thanks to Tokyo University for producing it.

Access: DBTBS

Constitutive promoters:

Use of basal transcription promoters, potentially the promoter for one or various rRNAs, P3 promoter[7], RNAP subunit gene promoters and metabolic gene promoters.

Annotated B. subtilis genome

Metabolic Pathways of B. subtilis

Probably ideal to pick a few (say 5) and characterise in order to find relative levels for use

1 rRNA promoter

1+ Metabolic related promoter (potential inducibility)

1 RNAP subunit promoter

P3 promoter

Another basally transcribe sequence

All non-constitutive promoters should remain functional in B.subtilis though leaky (basal) transcription rate will however be different, the key promoter is the one at the start of the chain...

Inducible promoters will most likely be taken from whichever shuttle vectors we can obtain as these are shown to work in B. subtilis whereas those presently in the registry may be E. coli specific.

With luck we may find a repressable promoter however that would just be the icing on the cake!

There is a large database of B.subtilis genes, promoters and terminators online that may prove extermely useful for locating promoters

DBTBS database

For example, the amyE gene regulator is repressable and so may be very useful amyE regulon

Promoters

Promoter	Type	Likely Promoter Strength	Binding Site(s)	Gene normally transcribed	Special features/other
prpmH	Constitutive	Weak	σ34 Χ 2	Ribosomal subunit L34	2 binding sites for same factor
prpoB	Constitutive	Basal	σ34	RNAP β subunit	None
pctc	Inducable	Basal	σ37	Ribosomal Subunit L25	σ34 also reads through this sequence, σ37 activated causing general stress response
pxyl	Repressed-Inducible	Weak-Strong	σ34, XylR	XylA, XylB etc.	Represser by XylR when not in presence of Xylose
pcomC	Consitutive-Inducable	Basal-Strong	σ34, ComK	Protein X	Has the normal basal transcriptional level but is up regulated by ComK. ComK levels in our system will be abnoramlly high due to the SinR repression of Rok (ComK repressor)
pstrong	Constitutive	Strong	σx	Protein X	XXXXX

NB. σ34 is the B.subtilis major factor, σ37 is the B.subtilis general stress response factor

Ribosome Binding Sites

On a preliminary basis, there appears to be some issues related to the B. subtilis RBS, particularly the sequence and also codon usage with the normal AUG start becoming UUG in at least some B. subtilis vectors[8]

The codon usage in B. subtilis indicates that UUG encodes leucine not methionine and as such the UUG methionine found on the B. subtilis vector[9], indicating the issue may be confined to just the start codon

In further support of B.subtilis utilising AUG as a start codon as is normal is the large amount of literature viewed in order to find suitable RBS sequences, of which many are associated within 5 - 15 nt of a start codon (and from the papers viewed, they were all AUG).

To clarify all. B.subtilis can use AUG, UUG amd GUG as a start codon[10]. It utilises the start codons preferentially, with AUG being more prefered and GUG being the rarely seen cousin.

Bioinformatics will be the order of the day, kudos to DBTBS again, along with NCBI and Uniprot...

3 RBS sequences have been determined using Bioinformatics (method to be described later) - RBS-sinR, RBS-comC and RBS-RbsR.

We are also designing our own RBS that may potentially be stronger than the natural ones (method also to be included later).

More improtantly, the B.subtilis RBS appears to be a 9bp sequence complementary to the a section of the 16S rRNA (3' end) from the 30S ribosome small subunit.[11] It is indicated that this may be due to a lack of the S1 protein in the B.subtilis ribosome.[12]. The complementary sequence is 5'-AAGGAGGTG-3'

As such, any sequence with high homology to this sequence may be used, in particular,Thorsted et al. (1999)[13] identified several putative RBSs in a B.subtilis vector that have reasonable homology. It may also be possible to simply use the complementary sequence to use as our RBS.

The distance between the RBS consensus and the start codon is the most critical factor in how effectively the RBS will function. In particular, it was found the 7bp was the optimum for high translation in B.subtilis for a consensus sequence[10]. It was also indicated that the sequence of surrounding bases had little impact.

Terminators

Investigation Required - we should just be able to use the biobricked E. coli ones but this'll need confirmation.

Motility

Vincent was kind enough to point out a paper detailing different methods (apart from flagellar-based propulsion) of movement in B. subtilis; it focusses mainly on "sliding" but mentions "flagella-dependent swimming and swarming, and flagella-independent, twitching, gliding, and sliding" as the known methods of movement. The paper is below.

• A defined medium to investigate sliding motility in a Bacillus subtilis flagella-less mutant

Seems by "sliding" they mean... being pushed outwards by the expansion of the colony. The bacteria facilitate this by secreting chemicals that lower the friction between themselves and the surface. This shouldn't interfere with our goal too much, as any bacteria outside the target zone should stop secreting (and start swimming) anyway.

epsE

B.subtilis has a copy of epsE in its genome and will express this if it encounters the specific signals. This would cause severe havoc in our system if the clutch was triggered randomly. As such it is necessary to turn epsE off. Previously we had considered using a ΔepseE strain, or knocking out epsE. However, upon inspection of the DBTBS database[1] it became clear that this was not required as it was naturally repressed by SinR. We can instead constitutively express SinR to turn of epsEΔ.

SinR is God!

Constitutive SinR production also has several useful side-effects. These include down-regulation of the alkaline protease subtilisin E, down-regulation of the extracellular protease ipa-15r, down regulation of Rok which will lead to upregulation of ComK (which increases the B.subtilis' competency) and halting the sporulation pathway by removing SipW and TasA from production.

These are all bonuses! It will also give us an example of cross-talk potentially helping the system if we use constitutive promoters that were previously attached to some rRNAs!

Protocols

Motility Assays

We'll need to run these to gain parameters for macro-modelling of the system. From the literature it seems there are two basic classes; plate-based assays involving characterisation of the over-night colony size and shape and cell-tracking based assays monitoring the activity of a single cell under a microscope (time-lapse microscopy).

Vincent, again, was kind enough to find some papers on motility assays for bacteria (the Methodology sections of the following papers are worth a look)...

1. Comparative Analysis of the Development of Swarming Communities of Bacillus subtilis 168 and a Natural Wild Type: Critical Effects of Surfactin and the Composition of the Medium
2. Swarming motility in undomesticated Bacillus subtilis
3. MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis
4. Properties of Motility in Bacillus subtilis Powered by the H+- coupled MotAB Flagellar Stator, Na+-coupled MotPS or Hybrid Stators MotAS or MotPB

...and also some software tools for carrying them out:

1. CellTrack: an open-source software for cell tracking and motility analysis
2. Automated measurement of cell motility and proliferation
3. Cell tracking software page
4. Cell Motion Tracking Software (Manual tracking)

http://www.openwetware.org/index.php?title=IGEM:IMPERIAL/2008/Bioprinter/Subteam_1&action=edit Editing IGEM:IMPERIAL/2008/Bioprinter/Subteam 1 - OpenWetWare

References

1. Sierro N, Makita Y, de Hoon M, and Nakai K. DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Res. 2008 Jan;36(Database issue):D93-6. DOI:10.1093/nar/gkm910 |
2. Nakamura LK, Roberts MS, and Cohan FM. Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov. Int J Syst Bacteriol. 1999 Jul;49 Pt 3:1211-5. DOI:10.1099/00207713-49-3-1211 |
3. Blair KM, Turner L, Winkelman JT, Berg HC, and Kearns DB. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science. 2008 Jun 20;320(5883):1636-8. DOI:10.1126/science.1157877 |
4. Härtl B, Wehrl W, Wiegert T, Homuth G, and Schumann W. Development of a new integration site within the Bacillus subtilis chromosome and construction of compatible expression cassettes. J Bacteriol. 2001 Apr;183(8):2696-9. DOI:10.1128/JB.183.8.2696-2699.2001 |
5. Zweers JC, Barák I, Becher D, Driessen AJ, Hecker M, Kontinen VP, Saller MJ, Vavrová L, and van Dijl JM. Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes. Microb Cell Fact. 2008 Apr 4;7:10. DOI:10.1186/1475-2859-7-10 |
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7. Leelakriangsak M and Zuber P. Transcription from the P3 promoter of the Bacillus subtilis spx gene is induced in response to disulfide stress. J Bacteriol. 2007 Mar;189(5):1727-35. DOI:10.1128/JB.01519-06 |
8. McLaughlin JR, Murray CL, and Rabinowitz JC. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283-91.
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