Part:BBa_K1362173:Experience

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UNIQd6a66d3d39d01754-partinfo-00000000-QINU UNIQd6a66d3d39d01754-partinfo-00000001-QINU

••••• Heidelberg 2014

Split sfGFP reconstitution This part was sucessfuly used by the Heidelberg iGEM team 2014 to reconstitute the fluorecence of split superfolder GFP by protein trans-splicing using the Npu DnaE intein. In the following the results of this experiment are shown. Figure 1: Cloning Strategy for the bicistronic expression of the two parts of split sfGFP. GFP_N was amplified from BBa_K1362173, CPEC overhangs and the NpuDnaE_N were in the PCR primer. NpuDnaE_C was amplified from BBa_K1362171 with Oligos containing CPEC overhangs and the GFP_C. Both PCR products were cloned in a biscistronic expression backbone using CPEC. Figure 2: Successful in vivo restorarion of sfGFP fluorescence. Fluorecence intensities detected at 475nm exitation and 512 nm emission wavelength for a period of 6 hours after induction. Split halves and splicing controls show no fluorescence. Simultaneous expression of the split parts leads to a strong increase of sfGFP fluorescence. Figure 1: Fused proteins result in fluorescence. A: Exemplary Gating of the sample. Front Scatter depicts the size, Site Scatter Granualarity of each counted event. B: Successful reconstituion of sfGFP after 4 h Figure 2: BL-21 (DE3) expressing non-splicing control in brightfiel channel (70ms exposure). E. Coli cells can be identified. Figure 3: BL-21 (DE3) expressing non-splicing control in Zeiss Standard GFP channel (1 s exposure, 480nm exitation, 505nm emission). Very little florescence is observed. Figure 6: BL-21 (DE3) expressing splicing contruct in brightfiel channel (70ms exposure). E. Coli cells can again be identified. Figure 7: BL-21 (DE3) expressing splicing in Zeiss Standard GFP channel (1 s exposure, 480nm exitation, 505nm emission). Formation of fluorescence is observed after protein splicing has taken place. To proof our idea of reactivating protein function by using inteins to fuse split parts, we decided to use fluorescent proteins. Fluorescent proteins are widely used, and the read out is simple. After expression of the sfGFP halves and split inteins, either as separated N- and C- terminal constructs or combined on one plasmid, the fluorescence was meassured via plate reader and FACS. After 4 hours of expression, a clear fluorescent signal is observed within in vivo samples with both, the N - and C - terminal parts of sfGFP, showing that fluorescence can be restored by trans-splicing (Figure 1). Unfortunately, fluorescence can not be observed in in vitro samples, indicating that no splicing reaction took place. The in vivo fluorescent signal is stronger in the supernatant than in the pellet. However, it is not as strong as the sfGFP control. The fluorescent signal of reconstituted sfGFP is about 100-fold stronger then the negative controls, when only a single part of sfGFP, or a non-splicing variant is expressed. The mixture of N- and C- terminal construct in vitro after lysis of the cells revealed no fluorescence, indicating that no splicing reaction took place. However we were missing an essential control in this experiment. In this experiment, we could not show the in vivo fluorescent signal when both non-splicing parts of sfGFP are expressed. This control is required, since there is the possibility that both parts of the protein come in close proximity to each other and form the complete protein independently of a splicing event. In course of the experiments we focused on the read-out via FACS, since this approach promises a more precise evaluation of fluorescence in single cell resolution. Prior to each FACS experiment, the machine had to be adjusted. On one hand, the gain was adjusted to the strongest fluorescent sample, the positive control, and the correct subpopulation of events had to be selected, a process termed gating. The front scatter depicts the size of the event, the side scatter granualarity. A proper gating is mandatory to assure correct measuring of single bacteria, instead of aggregated bacteria or debris (Figure 2A). The FACS data is showing a clear spectral shift towards the green fluorescence range for the splicing construct compared to the non-splicing control (Figure 2B). Likewise the single halves do not show fluorescence on their own. However, after 4 hours of induction the splicing product does not reach the same amount of fluorescence as the sfGFP. Since the fluorescent signal in both, the plate reader and the FACS, can be misleading, due to the possible reconstitution without splicing, we decided to verify our results by Western Blot. Prior to blotting, the proteins are denaturated. Thus, non-spliced sfGFP would appear as two separate parts on the blot, whereas irreversibly spliced sfGFP would appear as a single band. We could detect the single sfGFP parts, as well as the reconstituted sfGFP by the attached HIS - Tag. The Western Blot of the expressed constructs shows a significant amount of sfGFP reconstitution. Neither of the split intein halves reconstitutes sfGFP, only when both intein halves are expressed there is a sfGFP signal. Upon reconstitution, the splicing product is forming, which is only visible when restoration occurs. Interestingly, while there is still a strong band at the size of the NpuDnaEc-sfGFPc (C), there is no sfGFPN-NpuDnaEN (N) observable, indicating that the N - part is the limiting factor in the splicing reation. Considering the previous experiments, we assume that there is only an irreversible reconstitution when both, the N - and C - part are expressed, and that the meassured fluorescence is based on the splicing reaction.