Revision as of 16:47, 25 September 2013

L-form switch BioBrick

This BioBrick can be used as a switch to enable the model Gram-positive bacteria Bacillus subtilis to switch between cell-walled rod form and wall-less L-form. The BioBrick contains homologous regions to the pbpB and murE genes to allow integration into the chromosome of B. subtilis via homologous recombination. Within the BioBrick there is a xylose-controlled promoter PxylR. This promoter is upstream of the murE coding sequence when integrated into the chromosome of B. subtilis, placing murE expression under the control of PxylR. The murE gene is responsible for the synthesis of enzymes involved in production of peptidoglycan synthesis. Disruption of this pathway can be utilised to down-regulate production of the cell wall.

After integration of the BioBrick, the expression of murE is controlled by the presence, or absence, of xylose (via the PxylR promoter). When xylose is present, murE is expressed and functional cell wall is produced. When xylose is not present the cell wall is no longer produced and cells can transition to the cell wall-less L-form phenotype.

The BioBrick also contains a cat gene, conferring chloramphenicol resistance. This can be used to select for transformants in rod form.

This BioBrick only functions in enabling switching between walled cells and wall-deficient L-forms in B. subtilis.

To find out more about L-forms please visit our [http://2013.igem.org/Team:Newcastle/Project/L_forms L-forms] page.

Construction

700px Figure 1. Showing all of the components of L-form Switch Brick starting from the pbpb homology region at the far left, Spacer sequence, Chloramphenicol resistance marker, another Spacer, Pxyl promoter, murE RBS and finally the murE homology region at the far right.

The designed BioBrick was synthesised by DNA synthesis company DNA 2.0. The components within the BioBrick were as follows: -

Parts:

1. Biobrick prefix – RFC[10] standard.
2. 309bp of the end of the pbpB coding sequence.
3. Spacer.
4. Reverse and complement of chloramphenicol acetyl-transferase (cat) coding sequence (including native ribosome binding site (RBS) and promoter).
5. PxylR promoter.
6. Spacer.
7. RBS binding site for murE.
8. Spacer.
9. 331bp of the start of murE coding sequence.
10. Biobrick suffix – RFC[10] standard.

The synthesised BioBrick was then inserted into the pSB1C3 plasmid - the only plasmid acceptable by iGEM for part submission.

Cloning and Integration

The pSB1C3 plasmid containing the L-form switch BioBrick returned from DNA 2.0 was amplified in E.coli. The [http://2013.igem.org/Team:Newcastle/Notebook/protocols#Escherichia_coli_Competent_Cell_Preparation E. coli competent cell preparation] and [http://2013.igem.org/Team:Newcastle/Notebook/protocols#Escherichia_coli_Transformation E. coli transformation] protocols were used to transform this plasmid into E. coli cells for cloning. Transformant cells were incubated in order to clone the transformed plasmids. Then the cloned plasmids were [http://2013.igem.org/Team:Newcastle/Notebook/protocols#QIAprep_Spin_Miniprep_Kit extracted] and [http://2013.igem.org/Team:Newcastle/Notebook/protocols#Bacillus_subtilis_168_Competent_cell_Prep_and_Transformation transformed] into B. subtilis. Integration of the BioBrick from the vector plasmid to the host chromosome was facilitated through homologous recombination with crossover events within the pbpB and murE genes.

Testing and Characterisation

In order to produce L-forms we transformed the switch BioBrick into Bacillus subtilis str.168 and plated them out onto the LB media containing 5ug/ml Chloramphenicol and 0.8% Xylose. As can be seen on Figure 1, there were colonies growing on both plate 1 (B. Subtilis str. 168 + (Switch BioBrick)) and plate 2 (positive control, B.subtilis str. 168 + pGFPrrnB) as plasmid pMutin4 confers chloramphenicol resistance. There were no colonies growing on Plate 3 which is B. subtilis str. 168 onto the same media (negative control) as B. subtilis str. 168 does not naturally have resistance to chloramphenicol. This transformation results suggests that the switch BioBrick has been transformed into B. subtilis str.168 successfully.

Plate 1: B. Subtilis str. 168 transformed with switch BioBrick.	Plate 2: B.subtilis str. 168 transformed with pGFPrrnB (positive control).
Plate 3: B. subtilis str. 168 no transformation (negative control).	Figure 1. Plates of B. Subtilis str. 168 transformed with switch BioBrick, pGFPrrnB (positive control) and water (negative control).

Even though colonies were found on Plate 1, this didn’t conclude that our BioBrick has integrated into the pbpb and murE homology region replacing the spoVD section with the chloramphenicol resistance cassete and the Pxyl promoter. So, in order to test that the transformants have actually taken up the switch BioBrick and integrated it into the homology region on the B. subtilis chromosomes, some colonies were picked from Plate 1 and innoculated into NB/MSM media with xylose concentration varying between 0% and 0.8% and incubated in 30oC for 56 hours. After this incubation the morphology of the cells was checked under the microscope (Figure 2). In the batch of B. Subtilis incubated without xylose, all of the cells appeared as L-forms and in 0.2% xylose the majority of the cells were L-forms with only occasional rods. In 0.4% and 0.5% xylose the majority of the cells were rods with only a couple of L-forms being spotted and 0.6% and 0.8% xylose all cells appeared as rods. The results of this experiment have been summarised into table and graph formats which is displayed on Table 1 and Graph 1. This result concludes that our switch BioBrick works as designed and as the model suggested it would.

O% xylose	0.2% xylose
0.4% xylose	0.5% xylose
0.6% xylose	0.8% xylose

Figure 2. This shows the appearance of B. subtilis cells transformed with our switch BioBrick after being innoculated into a 1:1 ration of NB and MSM media with a range of xylose concentrations and incubated in 30oC for 56 hours. The xylose concentrations in %(w/v) are denoted under each image.

Table 1. Number of L-forms and rod cells and ratio of L-forms to rod cells of B. Subtilis 168 containing our switch BioBrick in Different xylose concentrations after 56 hours Incubation

Graph 1. Ratio of L-forms to rod cells of B. Subtilis 168 containing our switch BioBrick in Different xylose concentrations after 56 hours Incubation

We also considered using Flow cytometry to sort between L-form sand rods cells over a period of 72 hours to give us more detailed and accurate results of when cells actually starts to turn into L-forms from rods in media with different xylose concentrations. However, due to the high viscosity of the media (NB/MSM) which contains sucrose that L-forms need to survive we couldn’t calibrate the Flow Cytometry to do the reading.

To further test the switch BioBrick, we also transformed it into B. subtilis BSB1. As can be seen on Figure 3, there were colonies growing on both Plate 4 (B. Subtilis BSB1 + (Switch BioBrick)) and also on Plate 5 (positive control, B.subtilis BSB1 + pGFPrrnB) as plasmid pMutin4 confers chloramphenicol resistance. There were no colonies growing on Plate 6 which was inoculated with B. subtilis BSB1 onto the same media (negative control)as B. subtilis BSB1 does not naturally have resistance to chloramphenicol. With these results, we can conclude that the switch BioBrick can be successfully transformed and integrated into both commonly used B. subtilis lab strains.

Plate 4: B. Subtilis BSB1 transformed with the switch BioBrick.	Plate 5: B. Subtilis BSB1 transformed with the pGFPrrnB (positive control).
Plate 6: B. Subtilis BSB1, not transformed (negative control).	Figure 3. Plates of B. Subtilis str. BSB1 transformed with switch BioBrick, pGFPrrnB (positive control) and water (negative control).

The images in figure 4 and 5 show the different morphology of B. subtilis str. 168 and BSB1 before the integration of the switch BioBrick and following 56 hours incubation in 0% Xylose NB/MSM media.

Figure 4. B. subtilis str. 168 prior to transformation with our switch BioBrick

B. subtilis str. 168 after to transformation with our switch BioBrick

Figure 5. B. subtilis str. BSB1 prior to transformation with our switch BioBrick

B. subtilis str. BSB1 after to transformation with our switch BioBrick

This method of generating L-forms is very time consuming, and follows the [http://2013.igem.org/Team:Newcastle/Notebook/protocols#L-form_Growth_Media Liquid media protocol]. Figure 6 and Video 1 show L-forms being generated from B. subtilis rods, a process taking 12 hours. The rod-shaped cells can be seen shortening and widening over time as peptidoglycan cell wall synthesis is down-regulated and insufficient amounts of cell wall are able to maintain the regular rod-form cell shape. This results in the eventual adoption of circular L-form shape (visible at 686.25 minutes in Figure 6). Cells that achieve this form are completely unbound by cell wall.

In order to speed things up you can use lysozyme to initially remove the cell wall as in our [http://2013.igem.org/Team:Newcastle/Notebook/protocols#Protoplasting_to_generate_L-form Lysozyme protoplasting protocol]. The L-forms, as explained in the L-form page, will still be able to grow and divide, as opposed to regular protoplasts.

Figure 6. Fluorescence microscopy showing B. subtilis rod cells turning into L-forms over a period of nearly 12 hours following the [http://2013.igem.org/Team:Newcastle/Notebook/protocols#L-form_Growth_Media Liquid media protocol.]

Video 1. Time-lapse showing the conversion of B. subtilis rod cells to L-forms over a period of nearly 12 hours following the [http://2013.igem.org/Team:Newcastle/Notebook/protocols#L-form_Growth_Media Liquid media protocol.]

References

[http://www.ncbi.nlm.nih.gov/pubmed/22122227 Domínguez-Cuevas P, Mercier R, Leaver M, Kawai Y, Errington J. (2012) The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus. Molecular Microbiology, 83, 52-66.]

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603455/ Errington J. (2013) L-form bacteria, cell walls and the origins of life. Open Biology, 3, 120143.]

[http://www.ncbi.nlm.nih.gov/pubmed/19212404 Leaver M., Dominguez-CuevasP., Coxhead J.M., Daniel R.A. and Errington J. (2009) Life without a wall or division machine in Bacillus subtilis. Nature, 457, 849-853.]

[http://www.ncbi.nlm.nih.gov/pubmed/23452849 Mercier R., Kawai Y. and Errington J. (2013) Excess membrane synthesis drives a primitive mode of cell proliferation. Cell, 152, 997-1007.]

[http://www.ncbi.nlm.nih.gov/pubmed/11849491 Walker R., Ferguson CM., 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.]