Cre-recombinase functionality assay
We assayed doubly-transformed E. coli. BL21(DE3) cells with the both the CRY2-CreN/CIB1-CreC construct and the reporter construct with loxP-flanked RFP (EforRED) followed by a kanamycin-resistance gene (NPTII) (below).
CRY2-CreN/CIB1-CreC and lox-flanked RFP/kanaymycin-resistance reporter constructs used to test functionality of the light-inducible Cre-recombinase system.
We expected that in the dark, the CRY2-CreN and CIB1-CreC fusion proteins would be disassociated in solution. In the reporter construct, RFP would be expressed with transcription terminating at the STOP codon. When activated by blue-light, we expected CRY2 and CIB1 to associate, bringing together the fused Cre-recombinase fragments. The reconstituted Cre-recombinase would recombine the loxP sites of the reporter construct, excising RFP and allowing expression of the kanamycin resistance gene.
After growing these cells up in LB-amp/chlor to an A600 of 0.3, we aliquoted 40uL of these cells to the middle of each test plate as a control with no induction by IPTG (to activate expression of both constructs) or blue-light (to activate association of the split Cre-recombinase).
We then induced expression of both constructs with 0.2mM IPTG at room temperature for 15 minutes under blue-light from a gel imaging system for different intervals as indicated. After the each time interval, 50uL of the culture was diluted in 1mL LB and then 50uL aliquots spotted on the antibiotic-supplemented plates (ampicillin, chloramphenicol and 1x, 2x or 5x kanamycin). BL21(DE3) cells not induced with IPTG (plates 1,3 and 5) and cells transformed only with the CRY2-CreN/CIB1-CreC plasmid (plates 7 and 8) growing on LB-chlor were used as controls.
RFP was observed on all plates after a few days growth in the fridge (not shown), indicating leaky expression of the reporter construct. Growth of cells in the absence of IPTG or blue-light induction comparable to induced cells indicated leaky read-through of the reporter construct, despite the STOP codon at the end of the RFP reporter gene. To try to decrease this read-through, we incorporated a second terminator sequence after the EforRED gene. However, we observed similar results upon assaying this new construct.
Functional assay of light-induced Cre-recombinase using RFP/kanamycin-resistance reporter constructs. Expression of CRY2/CIB1-Cre-recombinase and loxP-flanked RFP in double transformants was either uninduced or induced with 0.2mM IPTG and plated on a combination of ampicillin, chloramphenicol and different concentrations of kanamycin. Cells were plated after incubated in blue-light after an interval specified on plate 1. Control plates 7 and 8 contain single CRY2-CreN/CIB1-CreC plasmid transformants.
This is in contrast to the study by Kennedy et al. (2010), where they observed low levels of recombination of the reporter construct alone and equivalent levels with CRY2-CreN and CIB1-CreC in the dark (attributed to background levels of the reporter spontaneously recombining and not light-independent CRY2-CIB1 interaction). Blue-light exposed cells showed a 158-fold increase in reporter. We used different light pulse and incubation times to induce our cells with light, though the activity of the reporter construct in the absence of the CRY2/CIB1-Cre-recombinase construct and light induction indicates that that device may also be faulty.
We hypothesised that a layer of kanamycin-resistant cells growing at the surface of the plate created a barrier to the antibiotic, allowing growth of non-resistant (and non-recombined) cells on top of this layer. If this is the case, then background levels of recombination might not be as high as suggested by these plates. In applied systems such as our NAD biosynthesis system, low levels of read-through might not be highly detrimental to the system, although death of a proportion of cells may decrease the efficiency of the system.
REFERENCE
Kennedy, M. J., Hughes, R. M., Peteya, L. A., Schwartz, J. W., Ehlers, M. D. & Tucker, C. L. Rapid blue light induction of protein interactions in living cells. Nat. Methods. 7, 973-975 (2010).
