Improvements | NYCU-Taipei

Overview

The fluorescent proteins have been used as the reporter gene for their observability. Not only does it have the critical color, but also be easily used and well studied. However, it still has its cons. The fluorescent proteins are not able to indicate real-time expression for their construct stability. Since it could last for a while after its expression, it strongly interferes with the detection of the expression endpoint. To prevent interference, we designed the degradation system and improved the mCherry (BBa_J18932) by appending it with a degradation tag (BBa_K1051207) as the component for the system. This tag is the recognition sequence for the adaptor, SspB, which will then carry protein with this sequence to the protease. Therefore, by combining with the protease and specific promoter, the fluorescent can be turned on and down.

Fig. 1) Example for degradation system

For testing whether the tag works as expected, the mCherry with degradation tag is combined with glpABC promoter. The construct is transformed into DH5α, and tested with fluorescent intensity and OD.

Fig. 2) A schematic of our BioBrick construction

Experiment Results

Construct the mCherry with the Tag Under the glpABC Promoter

To prevent stop codon behind FP interfering with the function of the degradation tag, the point mutations are performed. Since the stop codon is located at the HindIII cutting site, the validation is performed with HindIII and BamHI enzyme digestion.

Fig. 3) The result for enzyme digestion confirmation of glpABC-mCherry mutation

Fig. 4) The expected result for enzyme digestion confirmation of glpABC-mCherry mutation

Tag is synthesized due to its short length, and mut-glpABC-mCherry is appended with EcoRI and HindIII cutting site by PCR
We successfully built our construct, and the validation is performed by BamHI and EcoRI. For more information, please visit our project Results page.

Fig. 5) The result for enzyme digestion confirmation of glpABC-mCherry with tag

Fig. 6) The expected result for enzyme digestion confirmation of glpABC-mCherry with tag

Measurement

The origin mCherry (BBa_J18932) and our improvement version are tested under the glpABC promoter. Both designs are constructed within the pSB1C3 backbone with BBa_B0034 as the RBS and BBa_B0015 as the terminator. We culture the bacteria for 17 hr in a 5ml LB+CM medium. The samples are then transferred to 50 ml LB+CM in the 125 ml flasks and cultured under 37 degrees. During the culture, the fluorescent intensity and OD₆₀₀ are measured hourly.

According to the figure, we can see that the glpABC-mCherry with the degradation tag has significantly lowered its fluorescent intensity. The data suggest the tag works correctly, which means this part is ready for the part of the degradation system in the future.