User:Pakpoom Subsoontorn/Notebook/Genetically Encoded Memory/2008/10/06: Difference between revisions

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* The genetic systems has two states: A, B, C. The state can be changed  
* The genetic systems has two states: A, B, C. The state can be changed  
upon the expression of two different intergrase/excisionase systems.
upon the expression of two different intergrase/excisionase systems.
* In state A, the cell has two pieces of DNA, the chromosomal DNA and  
* 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
the free circular plasmid. The chromosomal DNA has two different bacterial
  integration sites for integrase, attB-1 and attB-2. The plasmid also has
  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.
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 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  
the system from state-A to state-B. On the other hand, the expression of  
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state-C  
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.
 
* One drawback is that their structures are poorly known and there is no known excision system yet.
     *  These are some ideas about reversible genetic knockout
     *  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 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.

Revision as of 20:22, 6 October 2008

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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.

  • 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 


   *  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

  • Should we push the review article on Genetically Encoded Memory to general

audiences? or just leave it as a note among ourselves?

  • As we discussed earlier, there are 3-4 levels of the project: The application level (like engineering a yeast to report its age, or an in vivo directed evolution system), the grand-challenge level (like making 8-bit counter) and the specific experiment level. How should we manage our time?
  • Relating to the question above, should I start a deep-research on a particular system (say, lambda integrase)? or should I explore broader ranges of tools (other enzymes, recombination system etc.) for now?

Science Questions/ To-do list

  • 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

References


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