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Team:Newcastle/End of crack & signalling system
From 2010.igem.org
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End of crack cell-signalling system
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 including pathogenic bacteria such as propionibacteria, staphylococci, clostridia, enterococci and streptococci. Subtilin also double off as a quorum sensing molecule, therefore we will be using the subtilin-based cell signalling system to trigger calcium carbonate precipitation and filament cell formation once the bacteria have reached a sufficient density inside a crack. Our project consists of 3 different Biobricks, a subtilin sensing Biobrick, a subtilin production Biobrick and a subtilin immunity Biobrick (Figure 1).
Figure 1: Schematic diagram of subtilin cell signalling system in B. subtilis 168
Subtilin-sensing BioBrick
Figure 2: Subtilin-sensing Biobrick constructed by Team Newcastle 2008
The subtilin-sensing Biobrick consists of the spaRK genes under the spaRK promoter. SpaR is a 220 amino acid protein that regulates the downstream production of subtilin. 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]. SpaK is a histadine kinase protein 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.
Subtilin production Biobrick
The subtilin production Biobrick consists of the spaBCTS gene cluster under an oxygen limitation sensitive promoter. In low oxygen condition, similar to conditions down a crack, spaS will be expressed. The immature SpaS will then have to be modified and transported out of the cell by SpaBCT, an ABC transporter.
Subtilin Immunity BioBrick
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.
Cloning Strategy
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 will be selected for based on the inability of it hydrolysing starch. 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 ATCC6633
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 m 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: 1. Negative Control – B. subtilis 168 2. Positive Control – B. subtilis ATCC6633 3. 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 |
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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