Revision as of 16:28, 1 October 2013

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Project Overview

We have created a BioBrick which makes the model Gram positive bacteria Bacillus subtilis lose or regain its cell wall, on our demand, while still allowing it to grow and divide. B. subtilis without a cell wall is better in many different ways and should be used as a new chassis for use in Synthetic Biology.

All you need to start using an L-form chassis is a culture of Bacillus subtilis, our L-form switch BioBrick and a set of instructions from us. Bacteria which have lost their cell wall which are still able to grow and propagate are called L-forms, or as we prefer to call them, naked bacteria. There are loads of things that you can do with naked bacteria, we explored a few of them:

(One of the best facts about naked bacteria is that they are osmotically sensitive, meaning that they will lyse if they escape into the environment. This means that they can be used in non-contained environments.)

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Genome Shuffling

Fusion of bacteria is made significantly easier without a cell wall. This forces the fusants to reproduce sexually, where their genomes recombine, and this can be used in directed evolution. We have shown L-forms fusing, and the recombination of their genomes.

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Introduction and Detection of Naked Bacteria in Plants

L-forms have been shown to form symbiosis in plants. We’ve shown that the naked bacteria that we created using our switch BioBrick also form these associations. Plants with naked bacteria show increased resistance to fungus and they could be used to deliver useful compounds to the plant. This could give better plant yields, more nutritious plants and reduce the need for spraying of fertiliser, pesticides or other compounds.

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Shape Shifting

The loss of the cell wall leaves L-forms protected by only a cell membrane. The plasma membrane of l-forms is quite fluid. The advantage of this is that these cells would be able to adapt to shapes of various cracks and cavities, or will be able to "squeeze through" tiny channels and deliver cargo to hard-to reach targets.

We tested this hypothesis by injecting the naked bacteria into specially designed microfluidics chambers and observing their behaviour under the microscope.

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Modelling

We understand the importance of modelling in synthetic biology and have attempted to model as much as we were able. We constructed a model of membrane behaviour of L-forms, which included stress conditions, cell growth and boundaries provided by the chamber walls. We have modelled the biophysical mechanism of the L-form cell membrane fusion. We also modelled what effect the BioBricks we developed would have on B. subtilis.

This isn’t a finite list of what can be done with naked bacteria, there’s loads more that can be done! L-forms are currently used to discover novel antibiotics which don’t act on the cell wall. L-forms can teach us a great deal about how bacterial life has evolved, through acting as a model for a cell wall-less bacterial progenitor, and through being able to test the ease of induction of endosymbiosis in cell wall-less organisms (Mercier et al. 2013).

L-form bacteria can be used in any process which protoplasts are currently used for. Protoplasts are bacteria which have been chemically induced to lose their cell wall. They cannot however grow or divide (as L-forms can) and are not classified as being alive. L-forms can be used to transform bacteria which are recalcitrant to transformation (Chang and Cohen 1979).

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References

Walker R, Ferguson CMJ, Booth NA and Allan EJ (2002) The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of ‘Botrytis cinerea. Letters in Applied Microbiology, 34, 42-45.

Chang S and Cohen SN (1979)High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Molecular Genetics & Genomics, 168, 111–115.

Mercier R, Kawai Y and Errington J. (2013) Excess Membrane Synthesis Drives a Primitive Mode of Cell Proliferation, Cell, 152, 997–1007.

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-<!--{{Team:Newcastle}}
-=Project=
-We aim to investigate L-forms of the model Gram positive bacteria, ''Bacillus subtilis''. L-forms are bacterial cells that are able to propagate and grow without a cell wall in contrast to protoplasts. We propose that L-forms can be used as a novel chassis for synthetic biology.
-Furthermore, we are exploring some of the interesting applications that bacteria in L-form state provide. These include cell fusion and genome shuffling. It is significantly easier to fuse bacteria without cell walls interfering with the process. Fusion of L-forms is reasonably easy in the presence of PEG which will cause genetic recombination between fusants, forcing bacteria to reproduce sexually, and can be used for directed evolution.
-We are also looking at the physical shaping of bacteria using microfluidic technology and insertion of L-form bacteria into plants. L-forms have been found growing naturally in plants and have been postulated to provide benefits in plants.
-==Themes==
-===[https://2013.igem.org/Team:Newcastle/Project/shuffling_endosymbiosis Shuffling Recombination and Endosymbiosis]===
-Using L-form to perform genetic recombination has been shown to be 20X more efficient than Sexual PCR or any other current techniques. The advantages of using L-form are, it can grow, divide and re-integrate the cell form back. We are looking into two L-form fusion to potentially improve or generate novel functions; and try to create new bacteria strains via (cre-loxP system). Introducing foreign organisms (spores, smaller bacteria) into L-form and to see what happen to it, and triggering internal sporulation. We will also try to model the biophysical mechanism of the L-form cell membrane fusion.
-===[https://2013.igem.org/Team:Newcastle/Project/plants Introducing and detecting L-forms in Plants] ===
-<p>L-forms and plants can exist in a symbiotic relationship as plants provide an osmotically suitable environment. In return, L-forms can confer benefits to their host including reducing the rate of fungal infection. We plan to wash seedlings in a solution of GFP labelled L-forms, allowing the seedlings to take up the bacteria. We will then view our L-forms inside the plant using confocal microscopy.
-</p><p>
-In the future, L-forms could be engineered to supply nutrients to plants, potentially increasing crop yield in low fertility soil. L-forms are osmotically sensitive, giving the ethical advantage that they lyse if they escape from their host plant into the environment. Please click this box for more information.
-</p>
-===[https://2013.igem.org/Team:Newcastle/Project/shape_shifting Shape Shifting]===
-             <p>
-The loss of the cell wall leaves L-forms protected by only a cell membrane. The plasma membrane of l-forms is quite fluid and it's shape is governed by laws of physics, just like that of eukarytotic cells. The advantage of this is that cells would be able to adapt to shapes of various cracks and cavities, or will be able to "squeeze through" tiny channels and deliver cargo to a hard-to reach target.
-               </p>
-               <p>
-We will test this hypothesis by injecting the L-forms into specially designed microfluidics chambers and observe their behaviour under the microscope. Prior to the experiment we will attempt to construct a rough model of the membrane behaviour under stress conditions, which include cell growth and boundaries provided by the chamber walls.
-              </p>
-{{Team:Newcastle/Sponsors}}
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 {{Team:Newcastle}}
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 (One of the best facts about naked bacteria is that they are osmotically sensitive, meaning that they will lyse if they escape into the environment. This means that they can be used in non-contained environments.)
-[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]
+<ul><li>[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]</li></ul>
 ===[https://2013.igem.org/Team:Newcastle/Project/shuffling_endosymbiosis Genome Shuffling]===
 Fusion of bacteria is made significantly easier without a cell wall. This forces the fusants to reproduce sexually, where their genomes recombine, and this can be used in directed evolution. [https://2013.igem.org/Team:Newcastle/Project/shuffling_endosymbiosis We have shown L-forms fusing, and the recombination of their genomes].
-[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]
+<ul><li>[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]</li></ul>
 ===[https://2013.igem.org/Team:Newcastle/Project/plants Introduction and Detection of Naked Bacteria in Plants] ===
 L-forms have been shown to form symbiosis in plants.  We’ve shown that the naked bacteria that we created using our [https://2013.igem.org/Team:Newcastle/Parts/l_form_switch switch BioBrick] also [https://2013.igem.org/Team:Newcastle/Project/plants#Results form these associations]. Plants with naked bacteria show [https://2013.igem.org/Team:Newcastle/Project#References increased resistance to fungus] and they could be used to deliver useful compounds to the plant. This could give better plant yields, more nutritious plants and reduce the need for spraying of fertiliser, pesticides or other compounds.
-[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]
+<ul><li>[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]</li></ul>
 ===[https://2013.igem.org/Team:Newcastle/Project/shape_shifting Shape Shifting]===
@@ Line 61: / Line 27: @@
 We tested this hypothesis by injecting the naked bacteria into specially designed microfluidics chambers and observing their behaviour under the microscope.
-[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]
+<ul><li>[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]</li></ul>
 === [https://2013.igem.org/Team:Newcastle/Modelling/Introduction  Modelling]===
@@ Line 80: / Line 46: @@
 Mercier R, Kawai Y and Errington J. (2013) Excess Membrane Synthesis Drives a Primitive Mode of Cell Proliferation, ''Cell'', '''152''', 997–1007.
-[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]
+<ul><li>[https://2013.igem.org/Team:Newcastle/Project Click here to navigate to the top of the page]</li></ul>
 {{Team:Newcastle/Sponsors}}

Team:Newcastle/Project

From 2013.igem.org