Part:BBa_K4307003

Bxb1 integrase

Bxb1 integrase, adapted from Mycobacteriophage Bxb1, is capable of catalyzing site-specific recombination between two attachment sites (attP and attB) and inverting the DNA sequence flanked by these two opposing sites. Bxb1 integrase is a kind of serine integrase, and the advantage of this enzyme over other site-specific recombinases is that the recombination reaction driven by serine integrases is highly directional.

This time, we express Bxb1 integrase in bacteria and check its capability of induce inversion.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 180
  Illegal BamHI site found at 558
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 973
  Illegal AgeI site found at 1333
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of Bxb1 integrase.

Fluorescence assay was done to characterize the biobrick.

To ensure the inversion effect of Bxb1 integrase, we locate mCherry and EGFP upstream and downstream of OR1-OR2 promoter respectively.

The gene of Bxb1 integrase was integrated with pBAD promoter. The fluorescence signal of mCherry was recorded at 16th hour after induction with 1% (w/v) arabinose in this experiment. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell. All groups were carried out twice to do a statistical analysis. Different experiments were induced in one 96 well plate. The culture temperature was 37℃ in this trial. .

Figure 2: Characterization of serine integrase function by fluorescence spectrophotometry.

qPCR assay was done to characterize the biobrick.

We applied quantitative RT-PCR to measure the inversion efficiency of serine integrase. The primers used in qPCR were designed to amplify sequences in between only after inversion caused by integrase. We analyzed the inversion efficiency of serine integrase by measuring the Ct values of qPCR before and after arabinose induction. The influence of bacterial amount on the Ct values of target gene was blanked by measuring the Ct value of a reference gene (resistance marker gene on the plasmid vector).

After intricate analysis, we summarized valid data and performed regression analysis to test our detection. (Table 1) Even though the exact inversion efficiency cannot be determined, relative magnitude demonstrated that our system induced a two-fold increase in the ratio of inversed promoter sequence (Figure 3).

Table 1: qPCR results of serine integrase characterization.

Induction temperature (℃)	Induction time (h)	Target sequence Ct (unit)	Reference sequence Ct (unit)
Group 1	Induced	32.9	32.18
	Not induced	35.07	31.72
Group 2	Induced	14.195	14.655
	Not induced	14.91	13.615
Group 3	Induced	14.905	15.28
	Not induced	14.975	15.025
Group 4	Induced	13.834	14.705
	Not induced	14.785	13.325
Regression analysis	Induced	- 0.8791 * p + q - 0.8961 = 0
	Not induced	- 0.9299 * p + q - 1.5752 = 0

Figure 3: Characterization of serine integrase inversion efficiency by RT-qPCR. An exemplary qP graph (left) and the regression results (right).

Conclusion

We successfully expressed the integrase from bacteriophage in bacteria, and the results of the fluorescence assay showed that integrase could have obvious inversion effect, but the change of Ct values shows that the action time of Bxb1 integrase was a little bit long.