Team:HKUST-Hong Kong/asm.html
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The E. trojan is a synthetic E.coli strain that is engineered to lack an essential gene, nadE, in its genomic DNA. To survive, this strain has to rely on a pre- introduced plasmid (pDummy) bearing the essential gene; thus forcing the bacteria to maintain the plasmid until an alternative source of nadE gene is present. The pDummy, however, has been designed to have a temperature- sensitive origin of replication which would cease to function if the bacterial cells are incubated under higher incubation temperatures (>42ᵒC???). <br><br> | The E. trojan is a synthetic E.coli strain that is engineered to lack an essential gene, nadE, in its genomic DNA. To survive, this strain has to rely on a pre- introduced plasmid (pDummy) bearing the essential gene; thus forcing the bacteria to maintain the plasmid until an alternative source of nadE gene is present. The pDummy, however, has been designed to have a temperature- sensitive origin of replication which would cease to function if the bacterial cells are incubated under higher incubation temperatures (>42ᵒC???). <br><br> | ||
For sub-cloning purposes, an E. trojan – compatible vector plasmid is designed. This carrier vector, like the pDummy, contains the nadE essential gene. Once a gene of interest is inserted into this vector, the plasmid can be transformed to the E. trojan for amplification. Incubating the transformed bacteria at a temperature high enough to inactivate the heat sensitive replication origin of the pDummy would result in pDummy loss, making it necessary for the cells to retain the insert- bearing pCarrier for survival. Bacterial cells that do not take up the pCarrier and its insert would be deprived of the nadE gene product and die; while those who do would survive and continue dividing. | For sub-cloning purposes, an E. trojan – compatible vector plasmid is designed. This carrier vector, like the pDummy, contains the nadE essential gene. Once a gene of interest is inserted into this vector, the plasmid can be transformed to the E. trojan for amplification. Incubating the transformed bacteria at a temperature high enough to inactivate the heat sensitive replication origin of the pDummy would result in pDummy loss, making it necessary for the cells to retain the insert- bearing pCarrier for survival. Bacterial cells that do not take up the pCarrier and its insert would be deprived of the nadE gene product and die; while those who do would survive and continue dividing. | ||
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4. Isolate recombinants<br> | 4. Isolate recombinants<br> | ||
5. Induce loss of pToolkit | 5. Induce loss of pToolkit | ||
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<h4 align=left><a name=component></a>1.3. Component details</h4> | <h4 align=left><a name=component></a>1.3. Component details</h4> | ||
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<b>iGEM 2010 Slovenia Split/FRET constructs</b><br> | <b>iGEM 2010 Slovenia Split/FRET constructs</b><br> | ||
- | The split CFP and YFP from the biobricks of Slovenia team last year were used as alternative reporters. The idea is to put one of the terminal fragments of a split fluorescence protein into the pDummy, and swap out the essential nadE gene from the genome with the other terminal fragment. Driven by pLac R0010, both fragments should express simultaneously when induced by IPTG and fluorescence signal would be observed as an indicator of successful recombination. | + | The split CFP and YFP from the biobricks of Slovenia team last year were used as alternative reporters. The idea is to put one of the terminal fragments of a split fluorescence protein into the pDummy, and swap out the essential nadE gene from the genome with the other terminal fragment. Driven by pLac R0010, both fragments should express simultaneously when induced by IPTG and fluorescence signal would be observed as an indicator of successful recombination.<a href=#top>[Top]</a> |
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Revision as of 13:59, 4 October 2011
1. ASM
1.1. Theory – how to select?
The E. trojan is a synthetic E.coli strain that is engineered to lack an essential gene, nadE, in its genomic DNA. To survive, this strain has to rely on a pre- introduced plasmid (pDummy) bearing the essential gene; thus forcing the bacteria to maintain the plasmid until an alternative source of nadE gene is present. The pDummy, however, has been designed to have a temperature- sensitive origin of replication which would cease to function if the bacterial cells are incubated under higher incubation temperatures (>42ᵒC???).
1.2. Method of assembly
To study the population dynamics and behavior of a certain antibiotics sensitive strain of E Coli in a medium of antibiotic, our E. Trojan that is introduced into the culture medium must not process a wide spectrum of antibiotic resistance that impose a selective advantage. At the same time, E. Trojan needs to be transformed with the T4MO gene to carry out its job of signal disruption.
Let’s consider the following scenario:
We would obtain three possible outcomes: Using this mutualistic relation, the desired pDummy can be maintained once the host bacterium develops an addiction it, and pToolkit can be lost in bacteria propagation if the expression of G can be shut off manually. Eventually, the bacteria not obtain any new antibiotic resistance genes but keep pDummy.
Development of addiction – use of the lambda RED recombination system Summary of construction flow:1. Assembly pDummy and pToolkit
1.3. Component details
Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)
oriR101 & repA101-ts is a set of low copy origin of replication derived from the pSC101 origin of replication. The repA101-ts gene codes for a heat-labile protein that is required in trans for the initiation of replication at oriR101. In our construct, our characterization has shown that plasmids with this origin of replication can only be maintained below than 300C, and partial maintenance of plasmid was observed within temperature range from 290C to 330C. This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by a nucleotide mutation.
split superfolder green fluroscent protein_split sfGFPsplit superfolder green fluroscent The sfGFPs are mutated variants of GFPs that has improved folding kinetics and resistance to chemical denaturants. Split sfGFPs at amino acid residues 214 and 215 have been reported to undergo spontaneous complementation to give green fluorescence. The two split constructs were produced from an existing biobrick – pBAD driven sfGFP BBa_I746908. CDS of sfGFP amino acid residues 1-214 were copied out for sfGFP1-10 using PCR and stop codon was added to the end. The sfGFP11 was produced in a similar fashion, with a start codon added to the front of the CDS of amino acid residues 215 to 238.
Essential gene nadE (BBa_K524003)
Replication initiator pi protein and ori-gamma from R6K plasmid
iGEM 2010 Slovenia Split/FRET constructs |
ASM1 ASM 1.1 Theory – how to select? 1.2 Method of assembly 1.3 Component details |
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