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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 180
Illegal BamHI site found at 558 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 973
Illegal AgeI site found at 1333 - 1000COMPATIBLE 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).
| 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.
NOTOC__
AttP & AttB
AttP and attB are two attachment sites of Bxb1 integrase, each of which is about 50 bp in length. Bxb1 integrase 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.
This time, we express Bxb1 integrase in bacteria and check its capability of induce inversion.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 24
Illegal BsaI site found at 55
Illegal BsaI.rc site found at 150
Characterization
The following figure demonstrates our successful construction.
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Figure 1: The construction results of AttP and AttB determined by sequencing.
Fluorescence assay was done to characterize the biobrick.
In order to test the compatibility of the sequence of two attachment sites we picked with Bxb1 integrase, we placed mCherry and EGFP upstream and downstream of OR1-OR2 promoter respectively.
We incorporated the gene of Bxb1 integrase with pBAD promoter. The fluorescence intensity of mCherry was recorded 16 hours after the E.coli strain was induced with 1% (w/v) arabinose in this trial. The OD600 was also measured in order to standardize the fluorescence signal per cell. Different experiments were induced in one 96 well plate and all samples were separated to two wells to get an average result. The OD600 and fluorescence signal was recorded in a plate reader after 16 hours induction at 37°C or 16°C.
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Figure 2: Characterization of serine integrase function by fluorescence spectrophotometry.
Conclusion
We demonstrated that the attP and attB we selected could be recognized by Integrase and trigger inversion, which laid the foundation for the design of logic gating.
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