Part:BBa_K4307006
attB-promoter-attP & Ser integrase
Serine integrase, adapted from bacteriophage, 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. We use Bxb1 integrase, an integrase found in Mycobacteriophage Bxb1 in our project.
This time, we combined attP and attB, the attachment sites of serine integrase with OR1-OR2, the part that has promoter function in the right operator (OR) of phage genome.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 361
Illegal PstI site found at 667 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 361
Illegal NheI site found at 350
Illegal PstI site found at 667 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 361
Illegal BglII site found at 525
Illegal BamHI site found at 289
Illegal BamHI site found at 549
Illegal BamHI site found at 927 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 361
Illegal PstI site found at 667 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 361
Illegal PstI site found at 667
Illegal NgoMIV site found at 4
Illegal NgoMIV site found at 1266
Illegal AgeI site found at 124 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 24
Illegal BsaI.rc site found at 1933
Illegal SapI site found at 106
Characterization
The following figure demonstrates our successful construction.
Figure 1: The construction results of attB-promoter-attP and Ser 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 action time was a little bit long.
| None |