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

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mini-Project: Synthetic Jumping DNA from integrase/excisionase systems

  • Motivation:
  • Inspired by TS Ham et al's double inversion switch, we would like to design other genetically encoded memory unit that function more efficiently. There are two major problem with TS Ham et al' system. First, the efficiency of invertase is low so most of the cells that are triggered to inverted their DNA don't do so. Second, the reaction by invertase is reversible so one can't

really have full control over the state of the system.

  • Bacteriophage's integrase/excisionase system can a promising tools for directional change in genetic code. In Nature, the expression of integrase alone cause attP site on viral DNA to insert into attB site on bacterial genome with high efficiency and specificity. The reaction by integrase itself is irreversible. However, when integrase and excisionase are present together, the process can be reverted, yielding the complete and separated viral genome and bacteria genome

system design and mechanism

  • 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

  • The inserted piece of DNA can removed by expressing excisionase, together with integrase. Thus, we could reverse state-B or state-C back to state-A again.
figure1
figure1

Challenges

  • Control the expression time/level of each integrase and excisionase
  • Efficiency and timing of insertion must be fast enough. Otherwise, there

is a possibility that we can loss the jumping piece of DNA

Questions

  • What're the rough time scales for integrase/excisionase system? esp comparing to cell cycle


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