Team:Hong Kong-CUHK/Parts

Rci site-specific recombinase

This part is responsible for the functioning protein - Rci site-specific recombinase (384-amino acid) in Shufflon system, a site specific DNA recombination would occur between two specific repeat sites.

With the expression of this gene, the DNA sequence would be shuffled randomly according to the specific repeat sites recognized by the recombinase.

The inversion frequency is affected by the length of flanking DNA sequence in vivo. Please refer to BBa_K361001

Usage and Biology

Usage:

• The Rci site-specific recombinase is utilized by the bacteria in order to adapt the environmental changes by rearranging their gene pattern, the DNA shuffling effect may provide selective advantages in the new environment to survive.

• Integrated into our system, the Rci site-specific recombinase now functions as an encryption system - shuffling information that are stored in DNA.

Biology:

• In vitro study of conditions affecting recombination:

1. Additions of magnesium ion, EDTA, or ATP had little effect on recombination →Neither an energy source nor divalent cations are necessary.
2. The inversion was sensitive to KCl concentration →Maximal inversion activity at a KCl concentration of 80 mM.
3. The inversion reaction showed a broad pH optimum between 7.6 and 8.5.
4. The inversion reaction has a higher reaction activity at 42 oC as compared with that of 25 ^oC and 30 ^oC.
5. Negatively supercoiled DNA substrate was required for Rci protein. Little to zero recombination was observed in relaxed or linear DNA.

• In vitro study of recombination:
  

About 15-20 % inversion / recombination

• Reference:

[1] Gyohda, A. & Komano, T. (2000). Purification and Characterization of the R64 Shufflon-Specific Recombinase. Journal of Bacteriology, 182 (10), 2787-2792.

[2] Gyohda, A., Zhu, S., Furuya, N. & Komano, T. (2005). Asymmetry of Shufflon-specific Recombination Sites in Plasmid R65 Inhibits Recombination between Direct sfx Sequences. The Journal of Biological Chemistry, 281 (30), 20772-20779.

Message gene template

This message system consists of a unique designated message sequence, being inserted with specific repeat sequence for recognition site by Rci site-specific recombinase.

• This message has 70 characters:

"We must learn to live together as brothers or perish together as tools"

• In a 4 bits system, the total sequence is 280 bp.

In theory, the sequence with length equal to/ lower than 536 bp has high inversion frequency after 50 generation.

Gyohda, A. & Funayama, N. (1997). Analysis of DNA Inversions in the Shufflon of Plasmid R64. American Society of Microbiology, 179 (6), 1867-1871.

Rci recombinase system

This is the protein genertors of Rci site-specific recombinase BBa_K361000, which is being linked with a promotor and a ribosome binding site in front of it, a double terminator after it.

Please refer to part   “Rci site-specific recombinase”

R0010:B0034 (lac promotor + RBS):

Construction intermediate of lac promotor and RBS (defined effiency =1)

K361000:B0014 (Rci + terminator)

Construction intermediate of BBa_K361000 and BBa_B0014 double bidirectional terminator