Difference between revisions of "Part:BBa K3882002"

Revision as of 01:50, 21 October 2021

Elimination of AHL by E. bsuahlterminator

What it is? Why it is necessary? Microorganisms often persist in their natural niches by attaching to surfaces and forming complex, sessile microbial communities known as biofilms. P. aeruginosa is one of the most common food pathogens which is able to form biofilms either independently by itself or jointly with other microorganisms. When the concentration of quorum sensing (QS) molecule, AHL, secreted by pathogens in the environment is above the threshold, P. aeruginosa will be coordinated by QS signal to construct the biofilms. AHL is the key signaling molecular and can initiate the bio-toxin production. Thus, we need a quick methods to detect the accumulation of AHL in contaminated food.

What it does? Our E. bsuahlterminator r bio-brick contains 2 key elements including PVDQ and eGFP. PVDQ is a protein coded by pvdq gene. The PVDQ gene is regulated by T7 promoter, Lac operator and T7 terminator. The PVDQ protein can catalyze the deacylation of acyl-homoserine lactone (AHL) which can prevent the activation of LUXR by eliminating the AHL in the environment. In order to increase the stability and show the production quality in a real time manner. We use a protein linker and 3x HA tag to link the PVDQ protein with eGFP. We also use 3xHis tag at the c-terminal of eGFP to increase the efficiency to purify the recombination proteins. Our results shows the great stability in solutions and almost no aggregation happens after elusion.

How to use it? E. bsuahlterminatorbio-brick can transform to any competent bacterial. We use E. Coli BL21 as a recipe and safe bacterial to do the transformation. We use the LB media to culture the E. bsuahlteminator. When the OD value reach to 0.8, we add 200 uM IPTG into the LB media to activate the E. bsuahlterminatorbio-brick inside at 30 degree centigrade for 4 hrs. After that, we use protein purification kit from Beyotime company, followed by detecting the concentration of PVDQGFP. The protein can be directly added into the LB media and spray on the surface of LB-agar plate as well as shrimp, meat. It has an inhibitory function on P. aeruginosa growth and can last for at least 5-8 hrs.

LB solid medium simulation

To further testify the inhibitory effect of PVDQ protein on P. aeruginosa, we also used LB solid medium to simulate food and cultured P.aeruginosa on LB plate. There are two groups: controlled group with only P. aeruginosa and experimental group with P. aeruginosa and PVDQ protein; each group has three plates.

Fig.1 LB solid medium simulation.

Fresh food test

To test the effect of PVDQ protein on P. aeruginosa in an actual environment, we brought fresh food to do this experiment. We have two groups: experimental group and control group with identical amounts of fish and shrimp respectively. Then we spayed 0.62 mg/ml PVDQ protein onto fresh food of the experimental group while did nothing to the control group. After putting those samples in a 4℃ refrigerator for six hours, we did agarose gel electrophoresis to test the amount of P. aeruginosa in each sample.

Fig.2 AGE result of fresh food samples.

The purification of PVDQ-GFP

The affinity chromatography column is made of high purified polypropylene, with a height of 66 mm and an inner diameter of 12.7 mm. The sieve plate is made of high purified ultra-high molecular weight polyethylene (UHWMPE); it has low adsorption, great hydrophilicity, and uniform aperture(about 50 microns). This tube can be used to fill Ni-NTA, and it is suitable for the separation and purification of proteins with His tag.

Fig.3 Affinity chromatography column.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]