Team:HKUST-Hong Kong/asm.html
From 2011.igem.org
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- | Here, we introduce a heat-sensitive origin of replication as the only origin of pDummy. When we intend to switch off the replication of pDummy, we can incubate EX at above | + | Here, we introduce a heat-sensitive origin of replication as the only origin of pDummy. When we intend to switch off the replication of pDummy, we can incubate EX at above 30°C. This origin would then cease to function, and pDummy cannot be maintained. Deprived of the essential gene and the corresponding vital product, EX cannot propagate, unless, it receives an alternative but heat insensitive analog of pDummy. <br><br> |
This analog, named pCarrier, is the essentially our vector in cloning. Under an unfavorably high temperature, only those EX that are transformed with the insert-bearing pCarrier will be able to propagate and survive, while the others cannot undergo division and are virtually eliminated from the population. Eventually, the pDummy can be considered to be "shuffled out" by pCarrier. Our designed selection system, in short, bases itself on plasmid shuffling, and thus eliminates involvement of antibiotic resistance genes in any of the cloning steps.<a href=#top>[Top]</a><br> | This analog, named pCarrier, is the essentially our vector in cloning. Under an unfavorably high temperature, only those EX that are transformed with the insert-bearing pCarrier will be able to propagate and survive, while the others cannot undergo division and are virtually eliminated from the population. Eventually, the pDummy can be considered to be "shuffled out" by pCarrier. Our designed selection system, in short, bases itself on plasmid shuffling, and thus eliminates involvement of antibiotic resistance genes in any of the cloning steps.<a href=#top>[Top]</a><br> | ||
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<b>4.3 Essential gene <i>nadE</i> (BBa_K524003)</b><br> | <b>4.3 Essential gene <i>nadE</i> (BBa_K524003)</b><br> | ||
- | <i>nadE</i> is a vital gene in E. | + | <i>nadE</i> is a vital gene in <i>E. coli</i>. It codes for NAD+ synthetase. In principle, removal of such gene from the genome would cause addiction of bacteria to a plasmid that has a copy of the gene. CyaR (a sRNA) regulates the expression of <i>nadE</i> post-transcriptionally. This feature is retained in our construct. Transcription of <i>nadE</i> operon requires the sigma-70 factor and is terminated by downstream extragenic sites. The <i>nadE</i> gene was cloned out from the genome of strain BL21(DE3), and was completed the <i>nadE</i> by having B0015 terminator assembled to its end. |
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<b>4.5 iGEM 2010 Slovenia Split/FRET constructs</b><br> | <b>4.5 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. <a href=#top>[Top]</a> | + | 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 <i>nadE</i> 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 02:15, 5 October 2011
STRAIN CONSTRUCTION
1. Constructing EX – the bacterial strain that allows selection without use of antibiotics
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.
2. How to select against EX without the vector plasmid? Our alternative selection method
Our EX will have one of its essential genes (genes that are required for viability) removed from its genome, and relocated onto an engineered plasmid pDummy. As illustrated, in order to survive, EX must rely on those extra-chromosomal copies of the essential gene; therefore, EX is addicted to pDummy. By having direct control over the replication of pDummy, we dictate the life and death of EX (and hence the name pDummy).
3. Stepping in the heart of construction - methods of assembly
3.1 Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system
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.
3.2 Development of addiction – use of the lambda RED recombination system
3.4 Summary of construction flow:
4. Details of the components – a closer look to the molecular basis of assembly
4.1 Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)
4.2 split superfolder green fluroscent protein_split sfGFP
4.3 Essential gene nadE (BBa_K524003)
4.4 Replication initiator pi protein encoded by pir gene (BBa_K524004) and ori-gamma from R6K plasmid
4.5 iGEM 2010 Slovenia Split/FRET constructs |
Strain Construction1. Constructing EX 2. How to Select? 3. Methods of Assembly 4. Component Details |
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