Part:BBa_K5206006

alsRm-Palsl-sfGFP

Use BBa_K5206005 to replace the aslR gene in BBa_K5206004 to screen for a better mutant biosensor.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3252
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2537
  

Results

（1）Plasmid construction

To construct alsR random mutant libraries, 25 μL of Mutazym, 2 μL of upstream and downstream primers (alsr - rtaq - FOR and alsR - rtaq - REV), different concentrations of manganese chloride (0.3-0.5 mM), and 10 ng of wild-type template DNA were added to the error-prone PCR reaction system, and the system was made up by adding ddH2O water.

Electrophoretic detection of PCR results

After ligation with the vector pCDFDuet-1-PalsI-sfGFP, which was also amplified using PCR, it was transferred into E. coli BL21 (DE3) and spread on LB agar plates. The plates were incubated overnight at 37℃ in an incubator for 12-16h, and single colonies on the plates were picked on the following day for initial validation .

Electrophoretic detection of PCR results

（2）Fluorescence detection

For colonies with correct band sizes, one group of each colony was incubated without D-allose and one group was added with a concentration of 20 mM D-allose in 24 deep well plates at 37℃, 650 rpm for 16-20 h. The fluorescence values of the solution at excitation wavelength 488 nm and emission wavelength 518 nm were measured with an enzyme marker after treatment of the samples. As shown, mutant 3 showed higher fluorescence intensity.

Electrophoretic detection of colony PCR

Fluorescence values of wild type and mutant at 20 mM D-allose concentration. 1:WT, 2-10:mutants

We carried out specificity assay and optimal temperature exploration for the mutant 3 biosensor. We cultured the engineered bacteria transferred into the mutant sensor with different concentrations of D-allose, D-glucose, D-allulose, D-fructose at different temperatures (20℃, 30℃, 37℃) and detected the expression of their fluorescent proteins.

The experiments showed that D-allose was relatively more responsive to the biosensor at 37℃, and lower concentrations of D-allose were able to respond with higher values of fluorescence intensity compared to the original biosensor. In addition, the mutated biosensor also has a relatively low response value to D-glucose, D-allulose, and D-fructose.

Effect of different concentrations of substrates on the biosensor at 37℃

Effect of different concentrations of substrates on the biosensor at 30℃

Effect of different concentrations of substrates on the biosensor at 20℃

BBa_K5206005 efficiently converts D-allulose to D-allose in a biocatalytic process, and the production of this conversion product can be accurately detected by an advanced biosensor system, thus providing a highly sensitive and selective analytical method for the biosynthesis of D-allose.

The BBa_K5206007 and the better-performing BBa_K5206005 were co-transformed into Escherichia coli BL21(DE3) to explore the effect of adding different concentrations of the substrate D-allulose on the biosensor at different temperatures.

One group of bacterial solution was always induced and cultured in a shaker at 30℃ for 18-24 hours. The other group was first induced in a shaker at 25℃ for 6-8 hours and then transferred to a shaker at 37℃ for continuous induction and culture.

The results showed that at 30℃, in the strain containing the sensor system, as the concentration of D-allulose increased, the fluorescence signal was enhanced in grades, proving the production of D-allose. When co-cultured at 25℃ and 37℃, when the concentration of D-allulose increased to 100 mM, the fluorescence signal slightly weakened.

Response of the biosensor to the addition of different concentrations of substrates at different temperatures