Team:Newcastle/End of crack & signalling system

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=End of crack cell-signalling system=
=End of crack cell-signalling system=
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Subtilin belong to a class of peptide antibiotics, called lantibiotic that contain polycyclic thioether amino acids as well as the unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid. It is produced by ''Bacillus subtilis'' ATCC 6633, which is effective against a wide range of Gram positive bacteria.  
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Subtilin belongs to a class of peptide antibiotics, called lantibiotic that contain polycyclic thioether amino acids as well as the unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid. It is produced by ''Bacillus subtilis'' ATCC 6633, which is effective against a wide range of Gram positive bacteria including pathogenic bacteria such as propionibacteria, staphylococci, clostridia, enterococci and streptococci.
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Subtilin also doubles up as a quorum sensing molecule, which we can use to trigger calcium carbonate precipitation and filament cell formation once the bacteria have reached a sufficient density inside a crack. We developed a subtilin immunity part and a subtilin production part to add to the already-existing [http://partsregistry.org/Part:BBa_K104001 subtilin sensing part on the parts registry].
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[[Image:Newcastle subtilin diagram.png|700px]]
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We aim to use a subtilin-based cell signalling system to trigger calcium carbonate precipitation and filament formation once our bacteria have reached a sufficient density inside a microcrack.  
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'''Figure 1:''' Schematic diagram of subtilin cell signalling system in ''B. subtilis'' 168
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Subtilin is a lantibiotic peptide produced naturally by ''Bacillus subtilis'' ATCC 6633 but not by ''Bacillus subtilis 168''. We aim to produce two BioBricks: one for production of subtilin, and, since subtilin is a lantibiotic, one for subtilin immunity. A subtilin-sensing BioBrick was produced by Newcastle's 2008 iGEM team.
 
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The ''spaIFEG'' gene cluster encodes two separate subtilin immunity systems, both present in ATCC 6633. Together they provide a high degree of subtilin resistance. 
 
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==Research==
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==Subtilin Production part==
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===2008 Subtilin-sensing BioBrick===
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[[Image:Subtilin_production.png|700px]]
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* A type 1 antimicrobial peptide [AMP] or lantibiotic produced by ''B. subtilis''
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**NB. Lantibiotic = peptide-derived antibiotics with high antimicrobial activity against various Gram-positive bacteria, including pathogenic bacteria such as propionibacteria, staphylococci, clostridia, enterococci and streptococci.
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**NB. Subtilin not same as Subtilisin!!!!!
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*Production of subtilin involves the ''spa gene cluster'' encompassing 10 genes, ''spaBTCSIFEGRK''
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[[Image:NCL - Biobrick for Subtilin Sensor.PNG]]
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The subtilin production Biobrick consists of the ''spaBCTS'' gene cluster under an oxygen limitation sensitive promoter. In low oxygen conditions, similar to conditions at the end of a crack with high cell density, ''spaS'' will be expressed. The immature SpaS will then have to be modified and transported out of the cell by SpaBCT, an ABC transporter.
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The construct above contains:
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We will place this part in front of the existing [http://partsregistry.org/Part:BBa_K104001 subtilin-sensor part], allowing the subtilin signal to propagate from the end of the crack throughout the cell population.
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*''spaRK promotor''
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*''spaR'' (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene usually regulates the downstream production of subtilin antibiotic. It has an N-terminal domain that can be phosphorylated and a C-terminal domian that has DNA binding properties [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0M-4D4XNMM-4&_user=224739&_coverDate=09%2F01%2F2004&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000014659&_version=1&_urlVersion=0&_userid=224739&md5=1f421f180a48e2d68b86da579cc7f920|1]
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*''spaK'' (subtilin peptide antibiotic Kinase) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of SpaR [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0M-4D5KTFV-1&_user=224739&_coverDate=09%2F01%2F2004&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000014659&_version=1&_urlVersion=0&_userid=224739&md5=8d450f455463638579798872811ae5c0|2]. This activates the DNA binding ability of the C-terminus of SpaR, which in turn initiates transcription of the downstream gene. In the case of our construct, this gene is ''gfp''.
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*''rrnO'' transcriptional terminator
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*''spaS promotor'' - a strong promotor inducible by upstream activation of spaRK. It can be placed in front any gene to regulate its activity.
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===Production and Immunity Bricks===
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This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302021 BBa_K302021 on the parts registry].
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*''spaBTCS'' genes are required for production.
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==Subtilin Immunity part==
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*''spaIFEG'' genes are required for immunity.
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*''spaB and C'' are modifiers of the subtilin precursor.
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*''spaT'' is a transporter.
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*''spaS'' is the gene coding for the subtilin precursor peptide.
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*''spaI'' 's gene product sequesters subtilin.
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*''spaFEG'' genes code for transporters.
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[[Image:spa.png]]
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[[Image:subtilin_immunity.png|700px]]
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Image from Modeling Subtilin production in ''Bacillus subtilis'' Using Stochastic Hybrid Systems. Hu et al.
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The subtilin immunity BioBrick consists of the ''spaIFEG'' gene cluster under a constitutive promoter, Pveg. Immunity is based on two systems. SpaI is a lipoprotein that will bind to and interacts with subtilin at the cell surface. SpaFEG forms an ABC transporter homologue that will expel subtilin from the cell membrane into the extra cellular matrix.
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==Coding Sequence==
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This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302018 BBa_K302018 on the parts registry].
==Cloning Strategy==
==Cloning Strategy==
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[[Media:Newcastle CloningStrategy_BioBrick_Subtilin_Immunity.pdf|Subtilin Immunity]]
[[Media:Newcastle CloningStrategy_BioBrick_Subtilin_Immunity.pdf|Subtilin Immunity]]
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==Subtilin Immunity BioBrick==
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==Testing and Characterisation==
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The ''spaIFEG'' gene cluster encodes two separate subtilin immunity systems, both present in ATCC 6633. Together they provide a high degree of subtilin resistance.
+
-
 
+
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[[Image:subtilin_immunity.png|700px]]
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-
 
+
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==Subtilin production BioBrick==
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[[Image:Subtilin_production.png|700px]]
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==Biochemical pathway==
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[[Image:subtilin.jpeg|600px]]
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==Computational Model==
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==Characterisation==
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===Testing and Characterisation===  
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====Selection for integration====
====Selection for integration====
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To select for integration of the plasmid into the chromosome, ''B. subtilis'' will be tested for the ability to hydrolyse starch. Integration of the BioBrick will be done by homologous recombination at the amyE gene, therefore destroying endogenous expression of amylase. Colonies that are not able to break down starch on agar plate will be selected and cultured for further test. Colonies that do not contain the integrated BioBrick will be able to hydrolyse starch, therefore forming a white halo around the colony as iodine interacts with starch to form blue colour.  
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To select for integration of the plasmid into the chromosome, ''B. subtilis'' will be tested for the ability to hydrolyse starch. Integration of the BioBrick will be done by homologous recombination at the ''amyE'' gene, therefore destroying the ability to express amylase. Colonies that do not contain the integrated BioBrick will be able to hydrolyse starch, therefore forming a white halo around the colony as iodine interacts with starch to form blue colour.
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====Isolation of subtilin from ''B. subtilis'' ATCC 6633====
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====Isolation of subtilin from ''B. subtilis'' ATCC6633====
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{|
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|1. ''B. subtilis'' ATCC6633 were grown in 250 ml flasks containing 100 ml of BHI broth and incubate for 48 h at 32°C in shaker at 125 cycles/min.  
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1. ''B. subtilis'' ATCC6633 were grown in 250 ml flasks containing 100 ml of BHI broth and incubate for 48 h at 32°C in shaker at 125 cycles/min.  
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|-
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2. Culture media were centrifuged at 10000g for 15 min and the supernatants were filtered through 0.22 m membrane.  
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|2. Culture media were centrifuged at 10000g for 15 min and the supernatants were filtered through 0.22 um membrane.  
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3. The pH of the filtrates has to be between 7.0 to 8.0 pH.
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|-
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4. Store the filtrates at -20°C for further use.
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|3. The pH of the filtrates has to be between 7.0 to 8.0 pH.
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|-
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|4. Store the filtrates at -20°C for further use.
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|}
===Characterisation of BioBrick===
===Characterisation of BioBrick===
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{|
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1. Make up 4 LB agar plates and inoculate with the following organism:
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|1. Make up 4 LB agar plates and inoculate with the following organism:Negative Control –'' B. subtilis'' 168,  Positive Control – ''B. subtilis'' ATCC6633 and Test (Duplicates) – ''B. subtilis'' 168 containing subtilin immunity BioBrick.
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1. Negative Control –'' B. subtilis'' 168  
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|-
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2. Positive Control – ''B. subtilis'' ATCC6633
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|2. Apply 20 ul of the cell free filtrate containing subtilin onto cellulose disks (6 mm) and place onto the LB agar plates.
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3. Test (Duplicates) – ''B. subtilis'' 168 containing subtilin immunity BioBrick
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|-
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2. Apply 20 l of the cell free filtrate containing subtilin onto cellulose disks (6 mm) and place onto the LB agar plates.
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|3. Plates were incubated for 24 h at 37 °C.
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3. Plates were incubated for 24 h at 37 °C.
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|-
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4. Zone of inhibition were then measured.
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|4. Zone of inhibition were then measured.
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|}
====Expected results====  
====Expected results====  
Line 107: Line 77:
A.S. Motta and A. Brandelli. (2002) Characterization of an antibacterial peptide produced by Brevibacterium linens. Journal of Applied Microbiology 92, 63-70.
A.S. Motta and A. Brandelli. (2002) Characterization of an antibacterial peptide produced by Brevibacterium linens. Journal of Applied Microbiology 92, 63-70.
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A.S. Motta, F. Cladera-Olivera and A. Brandelli (2004) Screen for antimicrobial activity among bacteria isolated from the amazon basin. Brazilian Journal of Microbiology 35, 307-310.
A.S. Motta, F. Cladera-Olivera and A. Brandelli (2004) Screen for antimicrobial activity among bacteria isolated from the amazon basin. Brazilian Journal of Microbiology 35, 307-310.

Latest revision as of 00:56, 26 October 2010

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Contents

End of crack cell-signalling system

Subtilin belongs to a class of peptide antibiotics, called lantibiotic that contain polycyclic thioether amino acids as well as the unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid. It is produced by Bacillus subtilis ATCC 6633, which is effective against a wide range of Gram positive bacteria including pathogenic bacteria such as propionibacteria, staphylococci, clostridia, enterococci and streptococci.

Subtilin also doubles up as a quorum sensing molecule, which we can use to trigger calcium carbonate precipitation and filament cell formation once the bacteria have reached a sufficient density inside a crack. We developed a subtilin immunity part and a subtilin production part to add to the already-existing [http://partsregistry.org/Part:BBa_K104001 subtilin sensing part on the parts registry].

Newcastle subtilin diagram.png

Figure 1: Schematic diagram of subtilin cell signalling system in B. subtilis 168


Subtilin Production part

Subtilin production.png

The subtilin production Biobrick consists of the spaBCTS gene cluster under an oxygen limitation sensitive promoter. In low oxygen conditions, similar to conditions at the end of a crack with high cell density, spaS will be expressed. The immature SpaS will then have to be modified and transported out of the cell by SpaBCT, an ABC transporter.

We will place this part in front of the existing [http://partsregistry.org/Part:BBa_K104001 subtilin-sensor part], allowing the subtilin signal to propagate from the end of the crack throughout the cell population.

This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302021 BBa_K302021 on the parts registry].

Subtilin Immunity part

Subtilin immunity.png

The subtilin immunity BioBrick consists of the spaIFEG gene cluster under a constitutive promoter, Pveg. Immunity is based on two systems. SpaI is a lipoprotein that will bind to and interacts with subtilin at the cell surface. SpaFEG forms an ABC transporter homologue that will expel subtilin from the cell membrane into the extra cellular matrix.

This is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K302018 BBa_K302018 on the parts registry].

Cloning Strategy

Subtilin Immunity

Testing and Characterisation

Selection for integration

To select for integration of the plasmid into the chromosome, B. subtilis will be tested for the ability to hydrolyse starch. Integration of the BioBrick will be done by homologous recombination at the amyE gene, therefore destroying the ability to express amylase. Colonies that do not contain the integrated BioBrick will be able to hydrolyse starch, therefore forming a white halo around the colony as iodine interacts with starch to form blue colour.

Isolation of subtilin from B. subtilis ATCC 6633

1. B. subtilis ATCC6633 were grown in 250 ml flasks containing 100 ml of BHI broth and incubate for 48 h at 32°C in shaker at 125 cycles/min.
2. Culture media were centrifuged at 10000g for 15 min and the supernatants were filtered through 0.22 um membrane.
3. The pH of the filtrates has to be between 7.0 to 8.0 pH.
4. Store the filtrates at -20°C for further use.

Characterisation of BioBrick

1. Make up 4 LB agar plates and inoculate with the following organism:Negative Control – B. subtilis 168, Positive Control – B. subtilis ATCC6633 and Test (Duplicates) – B. subtilis 168 containing subtilin immunity BioBrick.
2. Apply 20 ul of the cell free filtrate containing subtilin onto cellulose disks (6 mm) and place onto the LB agar plates.
3. Plates were incubated for 24 h at 37 °C.
4. Zone of inhibition were then measured.

Expected results

1. Negative control – Zone of inhibition to be observed
2. Positive control – Zone of inhibition not observed
3. Test (Duplicates) – Zone of inhibition not observed or a smaller zone of inhibition as compared to the negative control

Expected result.png

References

A.S. Motta and A. Brandelli. (2002) Characterization of an antibacterial peptide produced by Brevibacterium linens. Journal of Applied Microbiology 92, 63-70.

A.S. Motta, F. Cladera-Olivera and A. Brandelli (2004) Screen for antimicrobial activity among bacteria isolated from the amazon basin. Brazilian Journal of Microbiology 35, 307-310.


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