IGEM:IMPERIAL/2009/Encapsulation/Phase2/Colanic acid: Difference between revisions

Latest revision as of 08:12, 4 August 2009

Mucoid Encapsulation

[1]

Background

Colanic acid is an exopolysaccharide produced by many bacteria including E.coli.

It forms part of the cell surface slime and is required for the development of the three-dimensional structure and the depth of biofilms.

Since it is naturally produced by E.coli we can manipulate the pathway as opposed to building in a new one (e.g. alginate biosynthesis).

Colanic acid has been shown to offer production against acidic conditions and dessication, it is also non-pathogenic.

James Chappell 09:51, 23 July 2009 (EDT):Any evidence about how bacteria with this coat would survive through the GI track? Be great if there were some examples, obviously E.coli can survive to some extend as of course we can become infected by some strains but there maybe some more direct evidence out there.

James Field Response:

The following article shows that knocking out colanic acid synthesis decreases viability on acidic media and investigates exposure to bile salts. The paper also makes reference to the fact that the colanic acid capsule confers a strong negative charge which is thought to prevent the migration of protons into the cell. [2]

--SxE00 05:49, 24 July 2009 (EDT) I have a big concern here: isn't the purpose of the protection offered by the biofilm to keep enough of the cells alive so the colony may survive the temporariliy harsh conditions? But our cells will be dead, hence won't mutiply - the article does not seem to correct for cell growth, but I may be wrong. Worse still we may have destroyed the walls so the enzyme we will have manufactured will be only protected by the biofilm. Overall, the efficiency of our encapsulation will be far worse than the figures in the article. Please tell me I have missed something!

Matthieu (I know, the login is bizarre)

Structure

It is a polyanionic heteropolysaccharide containing a repeat unit with D-glucose, L-fucose, D-galactose, and D-glucuronate sugars that are nonstoichiometrically decorated with O-acetyl and pyruvate side chains.

Structure after carboxyl esterification:

Acid resistance

Colanic acid is shown to increase acid resistance of bacteria. Indeed, colanic acid biosynthesis is up-regulated during the storage of bacteria in acidic foods such as yogurt.[3]

Comparing between mutant E.coli O157:H7 and E.coli O157:H7 that produce colanic acid.

CA confers a strong negative charge to the cell surface which, we suggest may serve as a buffer by neutralizing protons at the cell surface, whereby preventing positively charged chemical groups from accumulating on cell envelopes and from penetrating into cells. We further hypothesize that the amount of CA on cell surfaces determines the buffering capacity of cells. When cells lose their ability to produce CA, cell surfaces become less negatively charged and thereby have reduced buffering capacity. When negatively charged cell surfaces are neutralized, protons will accumulate and enter cells freely. Such a change in intracellular pH will impair cell metabolism, causing cell death. [4]

--SxE00 09:16, 24 July 2009 (EDT)

your cells will be dead...

Membrane Tethering

Unlike other exopolysaccharides, colanic acid does not naturally bind to the cell surface but rather forms a thick mesh between cells. We must adjust colanic acid production to ensure that cells are encapsulated. It has recently been shown that E.coli K12 can link colanic acid polymers to the lipid A core.[5]

Interestingly, pathogenic strains of E.coli usually link their O-antigens to the lipid A core molecule but becuase the O-antigen is absent from E.coli K-12 (due to an IS5 insertion) it is possible to substitute in the colanic acid. This membrane tethered colanic acid is called Mlps.

O-Antigen:

Membrane Tethered Colanic Acid (Mlps):

Regulation

It should be noted that colanic acid production is upregulated in response to low temperatures, osmotic shock, dessication and blue light.[6]

Colanic acid is not produced under optimum conditions or inside a living host.

Colanic acid biosynthesis is facilitated by the wca (cps) gene cluster that contains 19 genes and is regulated by rcs phospho-relay system.

RcsB:

Upregulation of RcsB has been shown to induce colanic acid production.[7] Colanic acid production is also upregulated by blue light.[8]

We can decouple this pathway to control the thickness of our cellular capsule.

To efficiently link an input of blue light to an output of colanic acid, the circuit can be simplified by placing RcsB under the control of YcgE repressible promoter. This biobrick has been designed by the 2009 Leuven team (BBa_K238000). There is currently no BioBrick for RcsB although the sequence is known.

There are also RcsB knockout strains of E.coli which could be used to avoid cross-talk between pathways.

http://cgsc2.biology.yale.edu/Mutation.php?ID=77155

There was also a recent 2009 paper that used a RcsB knockout strain of E.coli. [9]

James Chappell 10:01, 23 July 2009 (EDT):Cool but the paper you have cited above shows that by contorlling the RscB you will effect your cell division so defiantly we need to consider this, is it okay to write quick summary of the paper and what unintended affects we might expect. Also it seemed from the diagram that RscB is really at a node to many different pathways, look for recent paper to see if they have filled out any of the downstream pathways more.

James Field Response: RcsB is indeed nodal and while this brings with it important considerations (e.g. cell division), it facilitates the activation of the ugd operon which is required for capsule synthesis. It also upregulates a number of acid tolerance proteins that might be advantageous. It is strongly agreed that more research needs to be done with respect to the global effect of RcsB.

It might also be interesting to put an amber stop codon in the RcsB gene and co-express SupD with our gene of interest. This way, capsule formation would be influenced by both a light input and the levels of the gene of interest. Having said this, you only have to look at mucoid colonies to see the excessive colanic acid production, this indicates that all cells within a high density population will become encapsulated (i.e. it will be difficult to efficiently relate the presence of a capsule to a discrete amount of protein).

Based on this, it might be better to add an inducer which initiates the production of a protein of interest at a known rate. After a certain period of time, a blue light is switched on which does two things:

1) Initiates encapsulation (& possibly trehalose synthesis).

2) Turns off the production of the protein of interest. This could be achieved by using blue light to trigger the expression of a repressor which binds to the promoter controlling the protein of interest. Of course this level of dosage control is dependent on our final application. For instance, if the xylanase/cellulase application were chosen, higher levels of protein would be advantageous and therefore it would not be necessary to repress synthesis upon encapsulation.

James Chappell 10:03, 23 July 2009 (EDT):I like this idea, but I feel that your trying to do the job that could automatically done by a timer.As a group I feel you need to review what options there are there for timers...I have not really seen any progress on this aspect.Also generally I would like you to think of decoupling this contorl/timer module from the biosynthesis module.

B3023:

A recent Nature paper has also shown that colanic acid capsule production can also be greatly upregulated via the expression of a putative transcription factor (B3023). [10]

Sequences

RcsB:

    DNA (648 bp)
       atgaacaacatgaacgtgattattgcggatgatcatccgattgtgctgtttggcattcgc
       aaaagcctggaacagattgaatgggtgaacgtggtgggcgaatttgaagatagcaccgcg
       ctgattaacaacctgccgaaactggatgcgcatgtgctgattaccgatctgagcatgccg
       ggcgataaatatggcgatggcattaccctgattaaatatattaaacgccattttccgagc
       ctgagcattattgtgctgaccatgaacaacaacccggcgattctgagcgcggtgctggat
       ctggatattgaaggcattgtgctgaaacagggcgcgccgaccgatctgccgaaagcgctg
       gcggcgctgcagaaaggcaaaaaatttaccccggaaagcgtgagccgcctgctggaaaaa
       attagcgcgggcggctatggcgataaacgcctgagcccgaaagaaagcgaagtgctgcgc
       ctgtttgcggaaggctttctggtgaccgaaattgcgaaaaaactgaaccgcagcattaaa
       accattagcagccagaaaaaaagcgcgatgatgaaactgggcgtggaaaacgatattgcg
       ctgctgaactatctgagcagcgtgaccctgagcccggcggataaagat

    Protein
       1 mnnmnviiad dhpivlfgir ksleqiewvn vvgefedsta linnlpklda hvlitdlsmp
      61 gdkygdgitl ikyikrhfps lsiivltmnn npailsavld ldiegivlkq gaptdlpkal
     121 aalqkgkkft pesvsrllek isaggygdkr lspkesevlr lfaegflvte iakklnrsik
     181 tissqkksam mklgvendia llnylssvtl spadkd

B3023:

    DNA (480bp)
       atgaccaacctgaccctggatgtgaacattattgattttccgagcattccggtggcgatg
       ctgccgcatcgctgcagcccggaactgctgaactatagcgtggcgaaatttattatgtgg
       cgcaaagaaaccggcctgagcccggtgaaccagagccagacctttggcgtggcgtgggat
       gatccggcgaccaccgcgccggaagcgtttcgctttgatatttgcggcagcgtgagcgaa
       ccgattccggataaccgctatggcgtgagcaacggcgaactgaccggcggccgctatgcg
       gtggcgcgccatgtgggcgaactggatgatattagccataccgtgtggggcattattcgc
       cattggctgccggcgagcggcgaaaaaatgcgcaaagcgccgattctgtttcattatacc
       aacctggcggaaggcgtgaccgaacagcgcctggaaaccgatgtgtatgtgccgctggcg

     Protein
       1 mtnltldvni idfpsipvam lphrcspell nysvakfimw rketglspvn qsqtfgvawd
      61 dpattapeaf rfdicgsvse pipdnrygvs ngeltggrya varhvgeldd ishtvwgiir
     121 hwlpasgekm rkapilfhyt nlaegvteqr letdvyvpla

Waal Ligase:

    DNA (1257bp)
       atgctgaccagctttaaactgcatagcctgaaaccgtataccctgaaaagcagcatgatt
       ctggaaattattacctatattctgtgcttttttagcatgattattgcgtttgtggataac
       acctttagcattaaaatttataacattaccgcgattgtgtgcctgctgagcctgattctg
       cgcggccgccaggaaaactataacattaaaaacctgattctgccgctgagcatttttctg
       attggcctgctggatctgatttggtatagcgcgtttaaagtggataacagcccgtttcgc
       gcgacctatcatagctatctgaacaccgcgaaaatttttatttttggcagctttattgtg
       tttctgaccctgaccagccagctgaaaagcaaaaaagaaagcgtgctgtataccctgtat
       agcctgagctttctgattgcgggctatgcgatgtatattaacagcattcatgaaaacgat
       cgcattagctttggcgtgggcaccgcgaccggcgcggcgtatagcaccatgctgattggc
       attgtgagcggcgtggcgattctgtataccaaaaaaaaccatccgtttctgtttctgctg
       aacagctgcgcggtgctgtatgtgctggcgctgacccagacccgcgcgaccctgctgctg
       tttccgattatttgcgtggcggcgctgattgcgtattataacaaaagcccgaaaaaattt
       accagcagcattgtgctgctgattgcgattctggcgagcattgtgattatttttaacaaa
       ccgattcagaaccgctataacgaagcgctgaacgatctgaacagctataccaacgcgaac
       agcgtgaccagcctgggcgcgcgcctggcgatgtatgaaattggcctgaacatttttatt
       aaaagcccgtttagctttcgcagcgcggaaagccgcgcggaaagcatgaacctgctggtg
       gcggaacataaccgcctgcgcggcgcgctggaatttagcaacgtgcatctgcataacgaa
       attattgaagcgggcagcctgaaaggcctgatgggcatttttagcaccctgtttctgtat
       tttagcctgttttatattgcgtataaaaaacgcgcgctgggcctgctgattctgaccctg
       ggcattgtgggcattggcctgagcgatgtgattatttgggcgcgcagcattccgattatt
       attattagcgcgattgtgctgctgctggtgattaacaaccgcaacaacaccattaac

     Protein
       1 mltsfklhsl kpytlkssmi leiityilcf fsmiiafvdn tfsikiynit aivcllslil
      61 rgrqenynik nlilplsifl iglldliwys afkvdnspfr atyhsylnta kififgsfiv
     121 fltltsqlks kkesvlytly slsfliagya myinsihend risfgvgtat gaaystmlig
     181 ivsgvailyt kknhpflfll nscavlyvla ltqtratlll fpiicvaali ayynkspkkf
     241 tssivlliai lasiviifnk piqnryneal ndlnsytnan svtslgarla myeiglnifi
     301 kspfsfrsae sraesmnllv aehnrlrgal efsnvhlhne iieagslkgl mgifstlfly
     361 fslfyiaykk ralglliltl givgiglsdv iiwarsipii iisaivlllv innrnntin