User:Pakpoom Subsoontorn/Notebook/Genetically Encoded Memory/2008/10/06: Difference between revisions
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==Project: Reversible Genetic Knockout== | ==mini-Project: Reversible Genetic Knockout== | ||
* The genetic systems has two states: A, B, C. The state can be changed upon the expression of two different intergrase/excisionase systems. | * The genetic systems has two states: A, B, C. The state can be changed | ||
In state A, the cell has two pieces of DNA, the chromosomal DNA and the free circular plasmid. | upon the expression of two different intergrase/excisionase systems. | ||
The chromosomal DNA has two different bacterial integration sites for integrase, attB-1 and attB-2. The plasmid also has two different phage integration | * In state A, the cell has two pieces of DNA, the chromosomal DNA and | ||
the free circular plasmid. The chromosomal DNA has two different bacterial | |||
integration sites for integrase, attB-1 and attB-2. The plasmid also has | |||
two different phage integration sites for integrase, attP-1 and attP-2. | |||
* The expression of the integrase-1 will insert attP-1 into attB-1, changing | |||
the system from state-A to state-B. On the other hand, the expression of | |||
integrase-2 will insert attP-2 into attB-2, changing the system from state-A to | |||
state-C | |||
* Φc31 (and other serine recombinase) seems to be good choices for modify DNA code in term of efficiency and simplicity. They need only ~30 sth recognition sites on the inserting and the target DNA; no host recombination factor seems to be required; it has been shown to function in vitro and in non-natural host ...like animal and plant cell. | * Φc31 (and other serine recombinase) seems to be good choices for modify DNA code in term of efficiency and simplicity. They need only ~30 sth recognition sites on the inserting and the target DNA; no host recombination factor seems to be required; it has been shown to function in vitro and in non-natural host ...like animal and plant cell. | ||
* One drawback is that their structures are poorly known and there is no known excision system yet. | * One drawback is that their structures are poorly known and there is no known excision system yet. |
Revision as of 20:21, 6 October 2008
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mini-Project: Reversible Genetic Knockout
upon the expression of two different intergrase/excisionase systems.
the free circular plasmid. The chromosomal DNA has two different bacterial integration sites for integrase, attB-1 and attB-2. The plasmid also has two different phage integration sites for integrase, attP-1 and attP-2.
the system from state-A to state-B. On the other hand, the expression of integrase-2 will insert attP-2 into attB-2, changing the system from state-A to state-C
* These are some ideas about reversible genetic knockout * Can we have a system of two pieces of DNA that can fuse and re-split? The fusion is triggered by signal-1, splitting is triggered by signal-2. Well, this will be similar to the way phages enter and exit their lysogenic cycle. Now, the challenges are the following. First, unlike natural phage, the split of DNA must not lead to the lytic cycle. We want to host to survive. Second, the host has to maintain the two separated pieces of DNA. Third, the fusion and splitting efficiency must be high. * Can we modify integrase system to function like transposon (but higher specificity/ efficiency)? The piece of jumping DNA of attP for two different integrase systems. Signal-1 makes it insert to attB-1; signal-1' cuts it out; signal-2 makes it insert at attB; signal-2' cut it out again. * Readmore about transgenic/ knockout organism. How to target a specific gene in a genome? Read Zebrafish knockout paper [edit] Management Questions/requests
audiences? or just leave it as a note among ourselves?
Science Questions/ To-do list
References |