Part:BBa_K1344002
Nuc-HlyA
This part is based on BBa_K729004, modified by deleting the stop codons. It's also fusion with HlyA secretional tag for extracelullar expression
Alpha-Amylase and Nuclease Characterization in Biofilm Removal
Background
Biofilm as matrix extracellular polymeric substances (EPS) causes an increase in antibiotic resistance and pathogenecity of pathogenic bacteria.
Result
- Produced an E. coli strain which can degrade biofilm matrix
- Demonstrated that E.coli can secrete amylase to hydrolyze amylum using iodine test on the supernatant
- Identified experimentally a potential of enzyme extracellular secretion into supernatant using HlyA tag to produce active enzyme.
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We read the absorbance of the biofilm assay by using ELISA Reader model 680 microplate reader in wavelength 595 nm. Then we make the data averages and performed into a diagram bellow:
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After calculate the average value of each treatment, we want to compare the data whether the data significantly different by using SPSS 16.00 analysis to find p value. Then we do data distribution analysis by using Saphiro-Wilk test.
From the Saphiro-Wilk analysis we find the p is less than 0.05, it is indicated that our data have normal distribution. After that, in order to support the hypothesis we continue to do Independent T-test analysis.
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Note: indicate the data were significant result (data show significantly different if p value < 0.05); red color indicate the most effective enzyme degrading biofilm matrix
MH : MalS-HlyA; SMH : S100-MalS-HlyA; NH: Nuc-HlyA; SRNH: S100-RBS-Nuc-HlyA
Mix: MalS-HlyA and S100-MalS-HlyA placed together in same LB broth
Discussion
Less absorbance value indicate more effective the action of enzyme degrade biofilm matrix. From the data above we focus on data strong constitutive promoter-RBS-MalS-HlyA and MalS-HlyA device. From data above show that α-amylase is more effective degrading Pseudomonas aeruginosa. α-amylase compared to no treatment shows significant result in degrading biofilm matrix (p<0.05). This is due to polysaccharides compound is greater in Pseudomonas aeruginosa. This data is consistent when the addition of α-amylase and nuclease together in LB broth, it is also show significant result in degrading biofilm matrix. But α-amylase less effective in degrading Vibrio cholera, Bacillus substilis, Staphylococcus aureus, Klebsiella pneumonia, and E.coli. We expect that the component of polysaccharides in those bacteria is lesser than in Pseudomonas aeruginosa. Besides that the mutant E.coli need more time to degrade the biofilm because we only incubate 5 hours 37oC. Another factor that make the data error is there were crystal violet left when crystal violet washing.
We compare second device, Strong constitutive promoter-RBS-Nuc-HlyA (SRNH) with no treatment of E.coli mutant, the result shows that, SRNH treatment is more effective in degrading Klebsiella pneumonia biofilm (p<0.05). We expect that extracellular DNA content in Klebisella biofilm is greater than others component. Comparation of mix and no treatment data show almost significant result in biofilm removal. (p=0.058). But we considered that data is significant.
We also can make a conclusion that no promoter and ribosomal RBS can not be recognized by RNA polymerases so the enzyme can not be expressed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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