Part:BBa_K2243033

TP901-1 attB_221F_TP901-1 attP

To test the influence of attBP sites of TP901-1 to terminator ECK120030221 (abbreviation: 221) in the forward direction.

Usage

We constructed this part to characterize the recombination efficiency of the recombinase Lactococcal phage TP901-1. It consists of the terminator ECK120030221 (abbreviation: 221) in the forward direction flanked by attB and attP sites of recombinase TP901-1. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is blocked.

Biology

The attP site of TP901-1 is used to integrate phage DNA at the host attB site of Lactococcal bacterium smegmatis, generating the recombinant junctions attL and attR. DNA cleavage and re-ligation occur at the central crossover region at attB and attP, which allows the sequence to be flipped, excised, or inserted between recognition sites. We obtained the terminator, attB and attP sites by oligo synthesis.

Characterization

We first characterized the terminator strength using the following formula:

Ts=〖GFP〗of the random sequence/〖[GFP]〗with the terminator

Terminator Strength Induced with 0.1mM IPTG

Terminator Strength Induced with 1mM IPTG

And we sifted out 6 desirable unidirectional terminators without potential cryptic promotor in both orientations.

Terminator Strength Induced with 0.1M IPTG

Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG

Terminator Reference Table

Media:Peking_trt.xlsx

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]