Team:GIFU TOKAI/Contribution
Characterization
Additional data of the part of the registry
BBa_K3222000
Description
BBa_K3222000 is an improved part of BBa_I746916 which encodes super folder GFP gene (Pedelacq et al. (2006): "Engineering and characterization of a super folder green fluorescent protein", Nature Biotech 24 (1) January 2006). We change 15A to G to remove Sap1 restriction site with the silent mutation. This part meets both Biobrick standard and Type2S standard now.
Why we chose BBa_I746916 as our target
From this year, iGEM community incorporates Type IIS standard. The past iGEM only permits Biobrick standard, and the past parts may include Bsa1 restriction site and Sap1 restriction site. In case of Type IIS standard, these sites are illegal. Even many teams use BBa_I746916 since it is quite useful when performing the quantitative experiment, these illegal sites may be tricky. Thus we decided to remove the sites by silent mutation. Changing 15A to G makes no changes in amino acid level. Both the codon GAG and GAA encodes Glu. Thus we re-analyzed BBa_I746916 to compare with our new part of sfGFP.
Methods
First, plasmid DNA of BBa_I746916 was amplified with Cont_For primer, Cont_Rev primer. The PCR product was re-amplified with T7 SD PRO primer and Cont_Rev primer. The PCR product was electrophoresed by 1.5% agarose gel and dyed by ethidium bromide for 20 minutes. After confirming the single band, the DNA was purified by FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan). Then, following the instruction of PUREfrex2.0 and, it expressed sfGFP. At this time, we used water as a negative control instead of template DNA. sfGFP expressed by the cell-free system was analyzed by its fluorescence and western blotting.
Results
In PURE system, BBa_I746916 was successfully expressed and, the expressed sfGFP was turned into green under the UV. The figure.1 shows the sfGFP under UV exposure. We also perfomred western blotting and confirmed the band of sfGFP around 27 kDa. Using Microplate Reader, we also performed the quntification of sfGFP. We used Fluorescein from the measurement kit to make calibration. The result is shown in the graph.
Figure.1 sfGFP under UV exposure (Left: BBa_I746916, Right: Negative control)
Figure.2 Western Blotting Analysis (Control: Water, Sample: BBa_I746916)
Figure.3 Fluorescein Calibration
Figure.4 Fluorescence quantification by Microplate Reader
Short Summary
The band was detected by Western Blotting. This part can be used as positive control for the cell-free system.
Please visit the link below to know about the part more. http://parts.igem.org/Part:BBa_I746916