Part:BBa_K3799002
Bovine pancreatic DNase I,serum endonuclease of Bos taurus
This part contain the coding sequence of BOvine pancreatic DNaseI.DNase I is a double-strand specific endonuclease that degrades DNA. Bovine pancreatic deoxyribonuclease I (DNase I) is a DNA minor grove-interacting nuclease, which shows relatively low specificity.Bovine pancreatic deoxyribonuclease I (bpDNase) cleaves double-stranded DNA with no sequence specificity making it suitable for degradation of bacterial biofilm.Deoxyribonuclease I (DNase I) enzymes cleave single or double-stranded DNA and require divalent metal ions to hydrolyze DNA yielding 3΄-hydroxyl and 5΄-phosphorylated products.
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]
Cloning and expression
Coding sequence of Bovine pancreatic DNaseI was obtained from TwistBioscience synthetically by adding Biobrick prefix and suffix.It was cloned into linearized PSB1C3 vector following normal biobrick assembly and transformed in Ecoli DH5α.Transformants were screened using colony PCR.
Due to time and space constraints for purification of the protein, characterization of DNaseI was carried out using industrially prepared DNaseI from Sigma Aldrich(D2025)
Characterization
We determined the activity of DNaseI on biofilm of Pseudomonas aeruginosa using 96 well plate biofilm assay.We did the assay at two different stages to determine the optimum concentration and time required for maximum degradation rate of bacterial biofilm.
Pretreatment of bacterial biofilm with DNaseI
Pretreatment was done to determine the optimum concentration of DNaseI which gives maximum degradation of Pseudomonas biofilm.Bacterial biofilm was grown in 96 well plates in medium containing DNaseI supplemented at different concentration ranging from 0-25 µg/ml for 72 hours.Biofilm was quantified by checking absorbance at 595 nm after Crystal violet staining.Biofilm degradation rate was compared by calculating biofilm percentage reduction which is given by
It was observed that at a concentration of 10 µg/ml gave maximum BPR.BPR didnt further increase with higher concentrations.This concentration was taken as a standard for further experiments.
Posttreatment of bacterial biofilm with DNaseI
Posttreatment was done to determine the optimum incubation time of DNaseI to get maximum degradation.Bacterial biofilm was grown in 96 well plates for 72 hours to get maximum yield.Following this,bacterial colonies were washed off and the pure biofilm in wells were treated with DNaseI supplemented in media at a concentration of 10 µg/ml.Different wells were incubated for time points ranging from 0-50 min.Biofilm yield per condition was quantified by measuring absorbance at 595 nm after crystal violet treatment.Biofilm percentage reduction was further determined to compare degradation rate at different time point.
Wells incubated for 40 min showed maximum biofilm degradation.
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