Difference between revisions of "Part:BBa K3716028"

Latest revision as of 01:37, 22 October 2021

CBD+gusA(E. coli)

Usage and Biology

This part contains a gusA sequence that expresses the β-glucuronidase and two CBD sequences which work as handle proteins to attach to cellulose.

Measurement iBowu-China 2021

Introduction

It is expected that this part will express a β-glucuronidase protein which is linked with two CBDs, i.e., cellulose binding domain. If the CBDs work as expected, then the expressed enzyme can be extracted and is expected to attach to cellulose added in the solution

Protocol

1. Transform the plasmids into E. coli BL21(DE3)
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37℃ in a shaker overnight.
3. SDS-PAGE.
   1. Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis.
   2. Add marker in the first lane on the left at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures.
4. Enzyme Activity
   1. Take 100 ul of bacterial solution, centrifuge at 10000g for 1min at room temperature and discard the supernatant. Resuspend with 100ul ddH2O, and add into this solution 100ul of standard test solution I (containing Phenolphthalein-β-D-glucuronide, and the test box was purchased from Nanjing Jiancheng Biotech company). Incubate the mixture at 37C for 1 hour.
   2. Observe the solution. pink or purple color indicates positive enzyme activity. Quantitatively the activity can be measured by OD540 reading on a microplate reader, divided by its OD600 reading for normalization of concentration.

Results

We used a pet28a plasmid and expressed this part with T7 promoter. The result below showed this part can have a successful expression in the E. coli strains, and the catalytic power of the β-glucuronidase enzyme can be positively identified.

Figure 2. Enzyme activity of β-glucuronidase-CBD. Left column: no iPTG added. Right column：0.05 mM iPTG added

Sequence and Features