Part:BBa_K3799003
Dnase I with promoter(R0010) , RBS(B0034), Terminator(B0015)
This part is used produce Bovine pancreatic DNaseI(BBa_K3799002) under IPTG inducible Ecoli promoter(BBa_R0010) aalong with RBS(BBa_B0034) and double terminator(BBa_B0015)
Usage and Biology
Bovine pancreatic 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
The coding sequence for DNaseI was initially found from NCBI and then procured synthetically adding biobrick prefix and suffix from TwistBioscience.Cloning was carried out following Normal biobrick assembly using combination of EcoRI and PstI.Linearized plasmid back bone of PSBIC3 obtained by PCR amplification using Plasmid specific primers and Gene fragment were digested with EcoRI and PstI and further ligated .The resultant plasmid was transformed into Ecoli DH5α.Transformed colonies were identified and further confirmed using colony PCR.
Primers used
Biobrick Forward- gatggaattcgcggccgcttctag, Biobrick reverse- gatgctgcagcggccgctactagta
Plasmid backbone forward- gctgcagtccggcaaaaaa, Plasmid backbone reverse- gtgaattccagaaatcatccttagcg
Sequencing forward- tgccacctgacgtctaagaa, Sequencing reverse- attaccgcctttgagtgagc
Due to time and space constraints for purification of 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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