Team:Hong Kong-CUHK/Parts

Part	Name	Type
BBa_K361000	Rci site-specific recombinase	Coding
BBa_K361001	Message gene template	DNA
BBa_K361011	Rci recombinase system	Generator
BBa_S04509	R0010:B0034 (lac promotor + RBS)	Intermediate
BBa_S04524	K361000:B0014 (Rci + terminator)	Intermediate

Rci site-specific recombinase

This part will prouce a 384-amino acid Rci site-specific recombinase used in shufflon system, which is a site-specific DNA recombination between two specific repeat sites as recognition.

After this recombinase is being expressed, it can alter the DNA sequence being inserted randomly with the specific repeat sites originally to become an unknown sequence.

This is a Wide type Rci recombinase.

• In the recombination activity study:

• Intramolecular inversion frequency = 0.062

• Intramolecular deletion frequency = 0 (recombinase inactive on this function)

• Intermolecular recombination = 0 (recombinase inactive on this function)

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

Usage and Biology

Usage:

• The Rci site-specific recombinase is used for the bacteria to adapt the change in environment by rearrange their gene pattern to create a new species of bacteria that has selective advantage in that changed environment for survival.

• The Rci site-specific recombinase is now exploited to randomly rearrange the DNA sequence carrying data. It allows data encryption to be possible.

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:

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

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.

Reference:

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