Team:HKUST-Hong Kong/asm.html
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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 300°C, and partial maintenance of plasmid was observed within temperature range from 290°C to 330°C. This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by a nucleotide mutation.<br> | 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 300°C, and partial maintenance of plasmid was observed within temperature range from 290°C to 330°C. This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by a nucleotide mutation.<br> | ||
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- | 4.2 split superfolder green fluroscent protein_split sfGFP<br> | + | <b>4.2 split superfolder green fluroscent protein_split sfGFP<br> |
sfGFP1-10 (BBa_K524001) [Twins: BBa_K524006]<br> | sfGFP1-10 (BBa_K524001) [Twins: BBa_K524006]<br> | ||
- | sfGFP11 (BBa_K524002) [Twins: BBa_K524007] | + | sfGFP11 (BBa_K524002) [Twins: BBa_K524007]</b><br> |
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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. | 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. | ||
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<img src="https://static.igem.org/mediawiki/2011/6/62/Ust_21.jpg" width=100 height=100><BR> | <img src="https://static.igem.org/mediawiki/2011/6/62/Ust_21.jpg" width=100 height=100><BR> | ||
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<a href=#constructing>1. Constructing EX</a> <br> | <a href=#constructing>1. Constructing EX</a> <br> | ||
<a href=#method>2. How to Select? </a><br> | <a href=#method>2. How to Select? </a><br> |
Revision as of 20:57, 4 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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