Team:Newcastle/Parts/l form switch

From 2013.igem.org

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{{Team:Newcastle}}
{{Team:Newcastle}}
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=Switch Brick=
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=Rod to L-form Switch BioBrick=
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==Purpose and justification==
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==Purpose and Justification==
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The purpose of this main ‘keystone’ biobrick is to facilitate the switching of ''Bacillus subtilis'' cells from rod-shape to L-form, wall-less cells.  Furthermore, this ‘switch’ should also enable L-form cells to return to rod-shape when required.
 
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This will be facilitated through the introduction of a xylose-controlled promoter (''PxylR'') upstream of the ''murE'' gene, which is involved in the biosynthesis of peptidoglycan.  The presence, or absence of a cell wall would be controlled through the presence, or absence of xylose, respectively.
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The purpose of this main ‘keystone’ BioBrick is to facilitate the switching of ''Bacillus subtilis'' cells from rod-shape to L-form, wall-less cells.  Furthermore, this ‘switch’ enables L-form cells to return to rod-shape when required.  This is facilitated through the introduction of a xylose-controlled promoter (''PxylR'') upstream of the ''murE'' gene, which is involved in the biosynthesis of peptidoglycan.  The presence, or absence of a cell wall can be controlled through the presence, or absence of xylose, respectively.  The antibiotic resistance marker chloramphenicol acetyl-transferase (''cat'') is upstream of this promoter region to allow for the selection of cells in which this system is integrated.
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The antibiotic resistance marker chloramphenicol acetyl-transferase (''cat'') is upstream of this promoter region to allow for the selection of cells, in which this system is integrated.
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Homologous recombination allows this BioBrick to be integrated into the chromosome of ''B. subtilis''.  The ''cat'' and ''PxylR'' sequences are flanked by sequences that are homologous with regions of the ''B. subtilis'' chromosome, which allows for insertion of the BioBrick at the intended loci.  ~300bp at the 5’ end of the BioBrick is homologous with the end of the ''pbpB'' gene.  Similarly ~300bp at the 3’ end of the biobrick is homologous with the start of the ''murE'' gene.
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Homologous recombination will allow this biobrick to be integrated into the chromosome of ''B. subtilis''.  The ''cat'' and ''PxylR'' sequences will be flanked by sequences that are homologous with regions of the ''B. subtilis'' chromosome, which will allow insertion at the intended loci.  ~300bp at the 5’ end of the biobrick will be homologous with the end of the ''pbpB'' geneSimilarly ~300bp at the 3’ end of the biobrick will be homologous with the start of the ''murE'' gene.
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Integration of the BioBrick facilitates xylose-mediated control over the expression of ''murE''.  This cascades through biosynthetic pathways to enable control over peptidoglycan synthesis and thus control over cell wall production.  Simply, in ''B. subtilis'' cells that have integrated the BioBrick into their chromosome, only in the presence of xylose is the cell wall producedWhen xylose is not present cells lose their cell wall and survivors adopt the L-form phenotype.
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==Modelling==
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The model of the L-form BioBrick [http://parts.igem.org/Part:BBa_K1185000 BBa_K1185000] can be found on the Registry of Standard Biological Parts.
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Modelling biosynthesis of peptidoglycan, specifically involving manipulation of the ''murE'' gene.
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<!--https://static.igem.org/mediawiki/2013/3/33/BareCillus_Switch.png-->
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Currently undergoing completion…
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==Construction==
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[[File:BareCillus_Switch.png|700px]]
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To be synthesised by DNA 2.0.
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Parts:
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==Design==
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1. Biobrick prefix – RFC 10 standard.
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2. 309bp of the end of the pbpB coding sequence.
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[[File:Switch 1.png|700px]]
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3. Spacer.
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''Figure 1. Showing all of the components of L-form switch BioBrick 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.''
 +
<br>
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4. Reverse and complement of chloramphenicol acetyl-transferase (cat) coding sequence (including native ribosome binding site (RBS) and promoter).
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The L-form switch BioBrick is based upon the regulatory sequence controlling expression of ''murE'' in ''B. subtilis'' LR2 - a strain capable of L-form state.  In this ''B. subtilis'' strain the peptidoglycan synthesis pathway required for the cell wall can be disrupted through control of ''murE'' expression - responsible for the synthesis of enzymes involved in peptidoglycan precursor biosynthesis.  A xylose-contolled ''PxylR'' promoter upstream of ''murE'' facilitates this control.  Upstream of this promoter the LR2 strain also contains a ''cat'' gene - conferring chloramphenicol resistance.  These genetic components differentiate the ''B. subtilis'' LR2 chromosome from that of ''B. subtilis'' 168.  On the LR2 chromosome these genetic components take the place of the ''spoVD'' gene (involved in sporulation) in ''B. subtilis'' 168.  It is these genetic elements that have been utilised to create a BioBrick that facilitates the switching of ''B. subtilis'' cells from rod state to L-form, and back again.  The flanking regions of the ''cat'' gene and the ''PxylR'' promoter - part of the ''pbpB'' coding sequence at the 5' end of this region of DNA and part of the ''murE'' coding sequence at the 3' end - are also of vital importance, allowing for integration of the BioBrick into the chromosome of ''B. subtilis'' via homologous recombination.
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5. PxylR promoter.
 
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6. Spacer.
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[[File:Switch.png|700px]]
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7. RBS binding site for murE.
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''Figure 2. Diagram showing how the L-form switch BioBrick is integrated into the B. subtilis chromosome.''
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8. Spacer.
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In order to package the genetic elements within the ''B. subtilis'' chromosome that enable this strain to transition from rod to L-form, the full sequence of the region between the ''pbpB'' and the ''murE'' genes had to be identified.  The sequence for the L-form switch region was obtained through a de novo assembly (using Sequencher 5.1 DNA sequencing software) of sequence data gained from sequencing between the ''pbpB'' and ''murE'' genes on the ''B. subtilis'' LR2 chromosome (using designed primers) with reads produced by high throughput sequencing of the full ''B. subtilis'' LR2 genome.  In particular, reads that did not assemble when using ''B. subtilis'' 168 as a reference genome were used in this assembly.  The primers that were designed to sequence the region between the ''pbpB'' and ''murE'' genes on the ''B. subtilis'' LR2 chromosome allowed sequencing from both the 5’ and the 3' ends of the desired sequence.  The design of the primers allowed for sequencing to include the final 309bp at the 3’ end of the ''pbpB'' coding sequence (upstream of the L-form switch region), as well as the sequence of the 331bp at the 5’ end of the ''murE'' coding sequence (downstream, and under the control, of the L-form switch).  Once fully sequenced, this region of DNA was able to be synthesised once compliant with the [http://parts.igem.org/Assembly_standard_10 BioBrick RFC[10]] standard.
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9. 331bp of the start of murE coding sequence.
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To allow the L-form switch BioBrick to be submitted to the iGEM registry of standard biological parts it needed to comply with BioBrick RFC[10] standard. In order to comply with this standard the BioBrick RFC[10] prefix and suffix sequences were added to the synthetic construct sequence using Sequencher 5.1 DNA sequencing software.  Two EcoRI restriction sites and a PstI restriction site that were present in the sequence were removed through single nucleotide changes using Gene Designer 2.0 - these restriction sites violated the terms of the RFC[10] standard.  Base changes were chosen to optimise the altered codons (in the open reading frame of coding sequences) for codon bias in ''B. subtilis''.  The altered genetic sequence was then able to be synthesised.
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10. Biobrick suffix – RFC 10 standard.
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==Modelling==
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Restriction map of biobrick (without biobrick prefix and suffix):
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To predict if the BioBrick we designed would function as desired, we modelled [https://2013.igem.org/Team:Newcastle/Modelling/L-form_Switch peptidoglycan biosynthesis] as peptidoglycan is the major component of the cell wall. The model suggested that peptidoglycan would not be  synthesised if ''murE'' was switched off.
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[[File:Clone Manager.png|500px]]
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Produced using Clone Manager 9 Professional Edition
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Cut sites include PstI and EcoRI (x2) – these are in the RFC 10 prefix and suffix restriction sites. The sequence will need to be altered to prevent being cut during excision of the biobrick.
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[[File:L-form biobrick.png|frameless|500px]]
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Produced using Gene Designer 2.0
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==Cloning and integration==
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'''1. The gene construct will be clone into pSB1C3, which is the only plasmid acceptable by iGEM for part''' submission.
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'''2. The plasmid that is returned from DNA2.0 will be amplified in E.coli. The following protocol outlines the procedure by which this will take place:'''
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===Competent cell preparation===
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Inoculate 300ml of LB broth with 1/20 volume of an overnight culture of the desired strain (e.g. 15ml overnight culture for 300ml of LB)
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• Grow the cells at 37°C (with shaking) to an absorbance at 600nm of 0.6
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• Chill the cells on ice and harvest by centrifugation at 4°C for 10minutes
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• Resuspend the pellet in 100ml of ice cold TFBI
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• Re-centrfuge the cells as before and resuspend in 20ml of ice cold TFBII
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• Aliqot 200µl volumes of the cell suspension into cooled, sterile microfuge tube, an flash freeze in an EtOH/dry ice bath
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• Store the cells at -80°C until requires
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===Transformation===
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• Thaw a 200µl aliquot of the desired strain of E.coli and add the transforming DNA. Incubate on ice for up to 30-45minutes.
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• Heat-shock the cells in a water bath for 120seconds, and place on ice again for 3-4 minutes
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• Add 1ml of LB broth and incubate the cells at 37°C for 1hour.
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• Spin down the cells in a microfuge (~20seconds) and remove the supernatant
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• Resuspend the cell pellet in ~200µl of broth or d.H2O and plate out on LB (agar at 1.5%), containing the appropriate selection markers.
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• Incubate plates overnight at desired temperature (normally 37°C)
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'''3. Transformed E.coli will be selected through growth on an antibiotic (Chloramphenicol) LB plate.'''
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'''4. Plasmid mini-prep procedure will then be performed in order to purify the plasmids.'''
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'''5. Then the plasmid will be digested with XbaI and SpeI., protocol as follow:'''
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- Using NEBuffer 2.1 at 37oC for 100% activity
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- To make up to 20µl of reaction mix
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- 15µl of DNA/plasmid
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- 1 µl of XbaI and 1 µl SpeI.
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- 2 µl of 10X NEBuffer 2.1 (must be diluted down to 1X)
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- 1 µl of water
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- Incubate the digestion mixture at 37oC for 3 hour
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'''6. 0.7% Agarose gel electrophoresis will then be used in order to separate the DNA fragments.'''
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'''7. Then, perform gel mediated DNA purification in order to purify the ‘switch’ gene construct (2,666bp).'''
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'''8. Prior to transforming this naked DNA (no plasmid), the B. subtilis 168 will be made competent through the following protocol:'''
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===Materials required for B. subtilis 168 transformation===
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• 10 µl of transformation DNA
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• Pipettes
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• 2X 15 ml falcon tubes
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• 2X 50 ml falcon tubes
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• Eppendorf tubes
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• Agar plates containing the appropriate antibiotic
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• Water bath
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• Centrifuge
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====SMM medium (1 litre) ====
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1. 2.0 g of ammonium sulphate
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2. 14.0 g of dipotassium hydrogen phosphate
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3. 6.0 g of potassium dihydrogen phosphate
 
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4. 1.0 g of sodium citrate dehydrate
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==Construction==
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5. 0.2 g of magnesium sulphate
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The designed BioBrick was synthesised by DNA synthesis company DNA 2.0. The components within the BioBrick were as follows: -
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6. Top up the rest of the medium with water
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Parts:
 +
#Biobrick prefix – RFC[10] standard.
 +
#309bp of the end of the ''pbpB'' coding sequence.
 +
#Spacer.
 +
#Reverse and complement of chloramphenicol acetyl-transferase (''cat'') coding sequence (including native ribosome binding site (RBS) and promoter).
 +
#''PxylR'' promoter.
 +
#Spacer.
 +
#RBS binding site for ''murE''.
 +
#Spacer.
 +
#331bp of the start of ''murE'' coding sequence.
 +
#Biobrick suffix – RFC[10] standard.
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====MM competence medium====
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The synthesised BioBrick was then inserted into the pSB1C3 plasmid - the only plasmid acceptable by iGEM for part submission.
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1. 10 ml of SMM medium
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==Cloning and Integration==
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2. 125 µl of Solution E (40% glucose)
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After the  pSB1C3 plasmid containing the L-form switch BioBrick was returned from DNA 2.0, it was amplified in ''E.coli''. The [https://2013.igem.org/Team:Newcastle/Notebook/protocols#Escherichia_coli_Competent_Cell_Preparation ''E. coli'' competent cell preparation] and [https://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 [https://2013.igem.org/Team:Newcastle/Notebook/protocols#QIAprep_Spin_Miniprep_Kit extracted] and [https://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.
-
   
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3. 100 µl of Tryptophane solution (at a concentration of 2 mg/ml
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4. 60 µl of Solution F (1M MgSO4)
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[[File:Switch.png|centre|700px]]
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5. 10 µl of 20% Casamino acids
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''Figure 2. Diagram showing how the L-form switch BioBrick is integrated into the B. subtilis chromosome.''
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6. 5 µl of 0.22% Fe-NH4-citrate
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<!--[[File:L-form biobrick construct.png|400px]]-->
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====Starvation medium====
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==Testing and Characterisation==
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1. 10 ml of SMM medium
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In order to produce L-forms we transformed the switch BioBrick into ''Bacillus subtilis'' str.168 and inoculated the transformants into LB media containing 5ug/ml Chloramphenicol and 0.8% Xylose. As can be seen on Figure 3, colonies grew 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 to ''B. subtilis''. There were no colonies growing on Plate 3 which was inoculated with ''B. subtilis'' str. 168 into the same media (negative control) as ''B. subtilis'' str. 168 does not naturally have resistance to chloramphenicol. These transformation results suggest that the switch BioBrick was transformed into ''B. subtilis'' str.168 successfully.
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2. 125 µl of Solution E (40% glucose)
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<html>
 +
<style type="text/css">
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#body img{
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    margin-bottom:3px;
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}
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table{
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border:2px solid black;
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margin-left:50px;
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margin-bottom: 10px;
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width: 650px;
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}
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.italic{
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font-style: italic;
 +
}
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table.borderless {
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border:0px solid white;
 +
}
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3. 60 µl of Solution F (1M MgSO4)
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</style>
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<body>
 +
<br>
 +
<table border="0">
 +
    <td><img src="https://static.igem.org/mediawiki/2013/d/df/BareCillus_168%2BSwitch.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 1: B. Subtilis str. 168 transformed with switch BioBrick.</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/5/54/BareCillus_168%2BrrnB.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 2: B.subtilis str. 168 transformed with pGFPrrnB (positive control).</div></td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/6/62/BareCillus_168%2BCm%2BH20.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 3: B. subtilis str. 168 no transformation (negative control).
 +
</div></td>
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    <td><div class="italic">Figure 3. Plates of B. Subtilis str. 168 transformed with switch BioBrick, pGFPrrnB (positive control) and water (negative control).</div></td>
 +
  </tr>
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</table>
 +
</body>
 +
</html>
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==Protocol==
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Even though colonies were found on Plate 1,  we couldn't conclude that our BioBrick had 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 had actually taken up the switch BioBrick and integrated it into the homology region in the ''B. subtilis'' chromosomes, some colonies were picked from Plate 1 and inoculated 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 4).  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. These results allow us to conclude that our switch BioBrick works as designed and as the model suggested it would.  
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This protocol will stretch for 2 days and aseptic techniques have to be applied for all steps.  
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===Day 1===
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<html>
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• Inoculate a single colony of Bacillus subtilis 168 into a 15 ml falcon tube containing 10 ml of MM competence media.  
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<body>
 +
<div id="tableWrap">
 +
<table border="0">
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/3/38/BareCillus_0.jpg" alt="Pulpit rock" width="268px" height="208px"><br><i>O% xylose</i></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/a/a6/BareCillus_0.2.jpg" alt="Pulpit rock" width="268px" height="208px"><br>0.2% xylose</td>
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/6/67/BareCillus_0.4.jpg" alt="Pulpit rock" width="268px" height="208px"><br>0.4% xylose</td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/c/cd/BareCillus_0.5.jpg" alt="Pulpit rock" width="268px" height="208px"><br>0.5% xylose
 +
  </tr>
 +
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/9/91/BareCillus_0.6.jpg" alt="Pulpit rock" width="268px" height="208px"><br>0.6% xylose</td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/2/25/BareCillus_0.8.jpg" alt="Pulpit rock" width="268px" height="208px"><br>0.8% xylose</td>
 +
  </tr>
 +
</table>
 +
</div>
 +
</body>
 +
</html>
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• For control, transfer 10 ml of MM competence media without the bacteria.  
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''Figure 4. This shows the appearance of B. subtilis cells transformed with our switch BioBrick after being inoculated 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.''
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• Incubate overnight in a shaking incubator at 37oC.  
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<table class="data">
 +
<tr>
 +
<th>%(w/v)xylose</th>
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                <th>Number of rods</th>
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                <th>Number of L-forms</th>
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                <th>Total cell number</th>
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                <th>% L-forms</th>
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                <th>% Rods</th>
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</tr>
 +
<tr class="odd">
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<td class="data">0</td>
 +
<td class="data">0</td>
 +
                <td class="data">16</td>
 +
                <td class="data">16</td>
 +
                <td class="data">100</td>
 +
                <td class="data">0</td>
 +
</tr>
 +
<tr class="even">
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<td class="data">0.2</td>
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<td class="data">1</td>
 +
                <td class="data">21</td>
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                <td class="data">22</td>
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                <td class="data">95</td>
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                <td class="data">5</td>
 +
</tr>
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<tr class="odd">
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<td class="data">0.4</td>
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<td class="data">26</td>
 +
                <td class="data">10</td>
 +
                <td class="data">36</td>
 +
                <td class="data">28</td>
 +
                <td class="data">72</td>
 +
</tr>
 +
<tr class="even">
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<td class="data">0.5</td>
 +
<td class="data">207</td>
 +
                <td class="data">3</td>
 +
                <td class="data">10</td>
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                <td class="data">1</td>
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                <td class="data">99</td>
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===Day 2===
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</tr>
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• Transfer 0.6 ml of the overnight culture into 50 ml falcon tube containing 10 ml of MM competence medium.
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<tr class="odd">
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• Incubate the tubes for 3 hours at 37oC.
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<td class="data">0.6</td>
 +
<td class="data">450</td>
 +
                <td class="data">0</td>
 +
                <td class="data">450</td>
 +
                <td class="data">0</td>
 +
                <td class="data">100</td>
 +
</tr>
 +
<tr class="even">
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<td class="data">0.8</td>
 +
<td class="data">480</td>
 +
                <td class="data">0</td>
 +
                <td class="data">480</td>
 +
                <td class="data">0</td>
 +
                <td class="data">100</td>
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• Warm up the starvation medium to 37oC in a water bath.
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</tr>
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• Add 10 ml of starvation medium (prewarmed) into each tube and incubate for a further 1-2 hours at 37oC.
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</table>
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• Transfer 0.4 ml of the medium not containing B. subtilis into one Eppendorf tube.
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• Add 10 µl of water into the tube.
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• Transfer 0.4 ml of the medium containing B. subtilis into two Eppendorf tubes.
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• Add 10 µl of DNA into one tube.
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• Add 10 µl of water into one tube.
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• Incubate the samples for 1 hour at 37oC in the shaking incubator.
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• The Eppendorf tubes have to be aerated, therefore incubate the tubes on their side.
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• Centrifuge the Eppendorf tubes at 13,000 rpm for 2 minutes.
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• Discard 0.3 ml of supernatant from each Eppendorf tubes.
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• Resuspend the pellet in the remaining supernatant and plate it onto agar plates containing the appropriate antibiotic.
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• Incubate the plates overnight at 37oC.
+
-
 
+
-
'''9. These competent cells will then be transformed with the naked DNA. The homologous region on the 5’ (pbpB) and 3’ (murE) will cause homologous recombination event. This Double cross over even will swap its native spoVD gene with the BioBrick.'''
+
-
 
+
-
[[File:L-form biobrick construct.png|500px]]
+
-
 
+
-
==Testing and Characterisation==
+
-
Transformants will be selected through growth on LB+Chloramphenicol+0.5-0.8% xylose media. Only those which took up the DNA and integrate the DNA will survive.
+
-
To characterise this BioBrick, the B. subtilis is to be grown on a media without xylose and then the morphology of the cells will be checked under the microscope. Successfully transformed B. subtilis cells should have converted to L-form state (lost their cell walls).  These are recognisable due to their rounded and non-uniform morphology. Cells may also vary greatly in size.
+
''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 ''
 +
[[File:Xylose conc.jpg|700px]]
-
There are 2 main approaches to do this:
+
''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''
-
====Liquid media====
+
We also considered using Flow cytometry to sort between L-forms and rods cells over a period of 72 hours to give us more detailed and accurate results regarding when cell 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.
-
• Mix equal volumes of 2xMSM and 2xNutrient Broth (Oxoid,NB) and add 5µg/ml of Chloramphenicol
+
To further test the switch BioBrick, we also transformed it into ''B. subtilis'' BSB1. As can be seen on Figure 5, 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.
-
• Add14-20mL of media to a 250ml Erlenmeyer flask/ 8-10 mL into 100 ml flask (to allow a large interface surface with air that will ensure proper oxygenation0
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• From the Xylose supplemented media, pick a few colonies and inoculate the media
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    <td><img src="https://static.igem.org/mediawiki/2013/3/3f/BareCillus_BSB1%2BSwitch.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 4: B. Subtilis BSB1 transformed with the switch BioBrick.</div></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/1/1f/BareCillus_BSB1%2Brrnb.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 5: B. Subtilis BSB1 transformed with the pGFPrrnB (positive control).</div></td>
 +
  </tr>
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  <tr>
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    <td><img src="https://static.igem.org/mediawiki/2013/a/ae/BareCillus_BSB1%2BCm%2BH20.jpg" alt="Pulpit rock" width="304" height="228"><div class="italic">Plate 6: B. Subtilis BSB1, not transformed (negative control).
 +
</div></td>
 +
    <td><div class="italic">Figure 5. Plates of B. Subtilis str. BSB1 transformed with switch BioBrick, pGFPrrnB (positive control) and water (negative control).</div></td>
 +
  </tr>
 +
</table>
 +
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-
• Gently mix the media to break up the cells
+
The images in figure 6 and 7 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.
-
• Incubate at 30oC without shaking
 
-
• Leave to grow for 1-2 days
 
-
Notes: A large media volume to flask volume ratio allows a thin layer of media which ensures diffusion of oxygen. Shaking is not necessary for growth, and if done should be very gently.
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    <td><img src="https://static.igem.org/mediawiki/2013/8/83/168_rod.jpg" alt="Pulpit rock" width="268px" height="208px"><br><i>Figure 6. B. subtilis str. 168 prior to transformation with our switch BioBrick</i></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/0/0e/168_L-form.jpg" alt="Pulpit rock" width="268px" height="208px"><br><i>B. subtilis str. 168 after to transformation with our switch BioBrick</i></td>
 +
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-
====Lysozyme protoplasting method====
+
<table border="0">
-
• Take the cells from Xylose supplemented plate and put it in 4mL of LB+xyl (0.8%) and incubate at 37oC for 2-3 hours
+
  <tr>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/2/2c/BSB1_rod.jpg" alt="Pulpit rock" width="268px" height="208px"><br><i>Figure 7. B. subtilis str. BSB1 prior to transformation with our switch BioBrick</i></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2013/4/45/BSB1_L-form.jpg" alt="Pulpit rock" width="268px" height="208px"><br><i>B. subtilis str. BSB1 after to transformation with our switch BioBrick</i></td>
 +
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 +
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• Dilute the cells from the 4mL media into the 10ml LB + 0.8% xylose media
+
This method of generating L-forms is very time consuming,  and follows the [https://2013.igem.org/Team:Newcastle/Notebook/protocols#L-form_Growth_Media Liquid media protocol]. Figure 8 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 there is insufficient amounts of cell wall 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 8).  Cells that achieve this form are completely unbound by cell wall.
-
• Check OD of cells every 1 hour and make sure it goes between 0.3-0.6 (mid exponential stage)
+
In order to speed things up you can use lysozyme to initially remove the cell wall as in our [https://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.
-
• Spin down @~5,500rpm for 4 minutes to remove the xylose
+
[[File:Rod-L.jpg|600px]]
-
• Wash once with LB
+
''Figure 8. Fluorescence microscopy showing B. subtilis rod cells turning into L-forms over a period of nearly 12 hours following the [https://2013.igem.org/Team:Newcastle/Notebook/protocols#L-form_Growth_Media Liquid media protocol.] ''
-
• Pellet the cells @4,500rpm for 15 minutes
 
-
• Resuspend pellet with Lysis solution (NB/MSM (2-4mL) + PenG (200µg/ml) + Lysozyme (2-4 mg/ml))
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</div>
 +
</html>
-
• Incubate for 1-1.30 hours @ 37oC for the lysozyme to work
+
''Video 1. Time-lapse showing the conversion of B. subtilis rod cells to L-forms over a period of nearly 12 hours following the [https://2013.igem.org/Team:Newcastle/Notebook/protocols#L-form_Growth_Media Liquid media protocol.] ''
-
• Check under microscope between the incubation period until 99.99% of cells are protoplast
+
==References==
-
• Spin down and re-suspend with NB/MSM  (10-15mL) + Chloramphenicol (5µg/ml) + 200µl of 200mg/ml PenG
+
[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.]
-
• Incubate @ 30oC for at least 2 days
+
[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.]
{{Team:Newcastle/Sponsors}}
{{Team:Newcastle/Sponsors}}

Latest revision as of 18:35, 28 October 2013

 
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Contents

Rod to L-form Switch BioBrick

Purpose and Justification

The purpose of this main ‘keystone’ BioBrick is to facilitate the switching of Bacillus subtilis cells from rod-shape to L-form, wall-less cells. Furthermore, this ‘switch’ enables L-form cells to return to rod-shape when required. This is facilitated through the introduction of a xylose-controlled promoter (PxylR) upstream of the murE gene, which is involved in the biosynthesis of peptidoglycan. The presence, or absence of a cell wall can be controlled through the presence, or absence of xylose, respectively. The antibiotic resistance marker chloramphenicol acetyl-transferase (cat) is upstream of this promoter region to allow for the selection of cells in which this system is integrated.

Homologous recombination allows this BioBrick to be integrated into the chromosome of B. subtilis. The cat and PxylR sequences are flanked by sequences that are homologous with regions of the B. subtilis chromosome, which allows for insertion of the BioBrick at the intended loci. ~300bp at the 5’ end of the BioBrick is homologous with the end of the pbpB gene. Similarly ~300bp at the 3’ end of the biobrick is homologous with the start of the murE gene.

Integration of the BioBrick facilitates xylose-mediated control over the expression of murE. This cascades through biosynthetic pathways to enable control over peptidoglycan synthesis and thus control over cell wall production. Simply, in B. subtilis cells that have integrated the BioBrick into their chromosome, only in the presence of xylose is the cell wall produced. When xylose is not present cells lose their cell wall and survivors adopt the L-form phenotype.

The model of the L-form BioBrick [http://parts.igem.org/Part:BBa_K1185000 BBa_K1185000] can be found on the Registry of Standard Biological Parts.


BareCillus Switch.png

Design

Switch 1.png

Figure 1. Showing all of the components of L-form switch BioBrick 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 L-form switch BioBrick is based upon the regulatory sequence controlling expression of murE in B. subtilis LR2 - a strain capable of L-form state. In this B. subtilis strain the peptidoglycan synthesis pathway required for the cell wall can be disrupted through control of murE expression - responsible for the synthesis of enzymes involved in peptidoglycan precursor biosynthesis. A xylose-contolled PxylR promoter upstream of murE facilitates this control. Upstream of this promoter the LR2 strain also contains a cat gene - conferring chloramphenicol resistance. These genetic components differentiate the B. subtilis LR2 chromosome from that of B. subtilis 168. On the LR2 chromosome these genetic components take the place of the spoVD gene (involved in sporulation) in B. subtilis 168. It is these genetic elements that have been utilised to create a BioBrick that facilitates the switching of B. subtilis cells from rod state to L-form, and back again. The flanking regions of the cat gene and the PxylR promoter - part of the pbpB coding sequence at the 5' end of this region of DNA and part of the murE coding sequence at the 3' end - are also of vital importance, allowing for integration of the BioBrick into the chromosome of B. subtilis via homologous recombination.


Switch.png

Figure 2. Diagram showing how the L-form switch BioBrick is integrated into the B. subtilis chromosome.

In order to package the genetic elements within the B. subtilis chromosome that enable this strain to transition from rod to L-form, the full sequence of the region between the pbpB and the murE genes had to be identified. The sequence for the L-form switch region was obtained through a de novo assembly (using Sequencher 5.1 DNA sequencing software) of sequence data gained from sequencing between the pbpB and murE genes on the B. subtilis LR2 chromosome (using designed primers) with reads produced by high throughput sequencing of the full B. subtilis LR2 genome. In particular, reads that did not assemble when using B. subtilis 168 as a reference genome were used in this assembly. The primers that were designed to sequence the region between the pbpB and murE genes on the B. subtilis LR2 chromosome allowed sequencing from both the 5’ and the 3' ends of the desired sequence. The design of the primers allowed for sequencing to include the final 309bp at the 3’ end of the pbpB coding sequence (upstream of the L-form switch region), as well as the sequence of the 331bp at the 5’ end of the murE coding sequence (downstream, and under the control, of the L-form switch). Once fully sequenced, this region of DNA was able to be synthesised once compliant with the [http://parts.igem.org/Assembly_standard_10 BioBrick RFC[10]] standard.

To allow the L-form switch BioBrick to be submitted to the iGEM registry of standard biological parts it needed to comply with BioBrick RFC[10] standard. In order to comply with this standard the BioBrick RFC[10] prefix and suffix sequences were added to the synthetic construct sequence using Sequencher 5.1 DNA sequencing software. Two EcoRI restriction sites and a PstI restriction site that were present in the sequence were removed through single nucleotide changes using Gene Designer 2.0 - these restriction sites violated the terms of the RFC[10] standard. Base changes were chosen to optimise the altered codons (in the open reading frame of coding sequences) for codon bias in B. subtilis. The altered genetic sequence was then able to be synthesised.

Modelling

To predict if the BioBrick we designed would function as desired, we modelled peptidoglycan biosynthesis as peptidoglycan is the major component of the cell wall. The model suggested that peptidoglycan would not be synthesised if murE was switched off.


Construction

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

After the pSB1C3 plasmid containing the L-form switch BioBrick was returned from DNA 2.0, it was amplified in E.coli. The E. coli competent cell preparation and 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 extracted and 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.

Switch.png

Figure 2. Diagram showing how the L-form switch BioBrick is integrated into the B. subtilis chromosome.


Testing and Characterisation

In order to produce L-forms we transformed the switch BioBrick into Bacillus subtilis str.168 and inoculated the transformants into LB media containing 5ug/ml Chloramphenicol and 0.8% Xylose. As can be seen on Figure 3, colonies grew 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 to B. subtilis. There were no colonies growing on Plate 3 which was inoculated with B. subtilis str. 168 into the same media (negative control) as B. subtilis str. 168 does not naturally have resistance to chloramphenicol. These transformation results suggest that the switch BioBrick was transformed into B. subtilis str.168 successfully.


Pulpit rock
Plate 1: B. Subtilis str. 168 transformed with switch BioBrick.
Pulpit rock
Plate 2: B.subtilis str. 168 transformed with pGFPrrnB (positive control).
Pulpit rock
Plate 3: B. subtilis str. 168 no transformation (negative control).
Figure 3. 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, we couldn't conclude that our BioBrick had 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 had actually taken up the switch BioBrick and integrated it into the homology region in the B. subtilis chromosomes, some colonies were picked from Plate 1 and inoculated 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 4). 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. These results allow us to conclude that our switch BioBrick works as designed and as the model suggested it would.

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

Figure 4. This shows the appearance of B. subtilis cells transformed with our switch BioBrick after being inoculated 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.

%(w/v)xylose Number of rods Number of L-forms Total cell number % L-forms % Rods
0 0 16 16 100 0
0.2 1 21 22 95 5
0.4 26 10 36 28 72
0.5 207 3 10 1 99
0.6 450 0 450 0 100
0.8 480 0 480 0 100

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

Xylose conc.jpg

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-forms and rods cells over a period of 72 hours to give us more detailed and accurate results regarding when cell 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 5, 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.


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

The images in figure 6 and 7 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.


Pulpit rock
Figure 6. B. subtilis str. 168 prior to transformation with our switch BioBrick
Pulpit rock
B. subtilis str. 168 after to transformation with our switch BioBrick
Pulpit rock
Figure 7. B. subtilis str. BSB1 prior to transformation with our switch BioBrick
Pulpit rock
B. subtilis str. BSB1 after to transformation with our switch BioBrick

This method of generating L-forms is very time consuming, and follows the Liquid media protocol. Figure 8 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 there is insufficient amounts of cell wall 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 8). 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 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.

Rod-L.jpg

Figure 8. Fluorescence microscopy showing B. subtilis rod cells turning into L-forms over a period of nearly 12 hours following the 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 Liquid media protocol.

References

[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.]

Newcastle University The Centre for Bacterial Cell Biology Newcastle Biomedicine The School of Computing Science The School of Computing Science