Coding
Part:BBa_K3519004
Designed by: Omkar Mohapatra Group: iGEM20_IISER-Tirupati_India (2020-10-07)
Revision as of 10:57, 24 October 2020 by Omkarmohapatra123 (Talk | contribs)
DNASEI
This part encodes the bovine pancreatic DNaseI, a highly potential and efficient endonuclease. Our use of this enzyme is to induce cellular death. The documentation below has been adapted from literature.
Usage and Biology
Sample Preparation of bp-cDNA:
Pancreatic tissues obtained from bovine were frozen in liquid nitrogen and stored at −70°C before processing for mRNA isolation. About 2 g of this deep-frozen pancreas was homogenized and the cellular total RNA was extracted by the guanidinium–phenol–chloroform method[1] . Next, using QuickPrep Micro mRNA Purification Kit, mRNA was isolated and was then used to synthesize cDNA with the help of CapFinder PCR cDNA Library Construction Kit.
PCR, cloning and DNA sequencing:
For PCR, the sample containing 10 pmol of various primers, 2.5 units of Dynazyme polymerase in 50 ml of 10 mM Tris–HCl (pH 8.8), 1.5 mM MgCl2, 0.1% Triton X-100 and 0.2mM dNTP was subjected to the first cycle (denaturation at 94°C for 3 min, annealing at 55°C for 3 min, extension at 72°C for 1min), the continued 35 cycles of amplification (denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min) and the final elongation step (10 min at 72°C) on a Thermal Cycler. The obtained PCR products were then purified by agarose gel electrophoresis and cloned into the pGEM-T Vector, and transformed into E. coli strain DH5a. The cDNA clones were sequenced with the T7 Sequencing Kit.
The forward 5∞-primer was synthesized based on the two conserved regions of human and rat DNase I cDNA sequences. The 3∞-fragment of the bp-DNase I cDNA was amplified by PCR with this forward 5∞-primer and an oligo (dT ) primer. The PCR products were cloned and sequenced. Based on the sequence of this 3∞-fragment, a specific reverse primer was synthesized and used with the CapSwitch oligonucleotide to amplify the 5∞- fragment of the bp-DNase I cDNA using PCR. The PCR products were again cloned and sequenced. The sequences of the 5∞- and 3∞-fragments showed overlaps and together covered the full-length of the coding region. The 1295bp cDNA sequence elucidated from the two fragments includes 120 bp of the 5∞-untranslated region, 846 bp of the reading frame and 329 bp of 3∞-untranslated region linked to a poly-A tail. A presumptive polyadenylation signal is present 29 bp upstream of the poly-A tail. The reading frame can be translated as a polypeptide chain of 282 aa, including a hydrophobic signal peptide of 22 aa and the polypeptide of bp-DNase I.
The forward 5∞-primer was synthesized based on the two conserved regions of human and rat DNase I cDNA sequences. The 3∞-fragment of the bp-DNase I cDNA was amplified by PCR with this forward 5∞-primer and an oligo (dT ) primer. The PCR products were cloned and sequenced. Based on the sequence of this 3∞-fragment, a specific reverse primer was synthesized and used with the CapSwitch oligonucleotide to amplify the 5∞- fragment of the bp-DNase I cDNA using PCR. The PCR products were again cloned and sequenced. The sequences of the 5∞- and 3∞-fragments showed overlaps and together covered the full-length of the coding region. The 1295bp cDNA sequence elucidated from the two fragments includes 120 bp of the 5∞-untranslated region, 846 bp of the reading frame and 329 bp of 3∞-untranslated region linked to a poly-A tail. A presumptive polyadenylation signal is present 29 bp upstream of the poly-A tail. The reading frame can be translated as a polypeptide chain of 282 aa, including a hydrophobic signal peptide of 22 aa and the polypeptide of bp-DNase I.
Expression of pETDNaseI:
The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. LB broth medium containing 0.1 mM IPTG was used for susceptibility testing. The susceptibility of E. coli BL21(DE3) harboring appropriate plasmid constructs was tested against sulfamethoxazole. For disk diffusion assay, E. coli BL21(DE3) harboring appropriate plasmid constructs was grown at 37 °C overnight in 5 mL of LB medium supplemented with 100mg/L of ampicillin. The overnight culture was transferred into fresh medium containing ampicillin and incubated at 37 °C up to an OD600 = 0.4. The bacterial suspension was spread on LB agar supplemented with ampicillin and IPTG (0.2 mM final concentration). Filter paper disks with sulfamethoxazole (20 μg) were overlaid onto the E. coli lawn and plates incubated at 30 °C overnight.
Colonies of E. coli strain BL21(DE3)pLysE transformed with pETDNaseI were cultured at 37°C in LB containing 50 mg of ampicillin/ml. IPTG induction (1mM) was doen when the absorbance of the culture medium (at 600 nm) reached approximately 1. After 3h of induction, centrifugation at 2000g (20min) was done to sediment the cells and cell debris. Due to the large amount of cell lysis after induction with IPTG, DNase I activities were found in both the cell pellet and supernatant fractions. The approximate yield from the induced culture was 3500 units/l as measured by the hyperchromicity assay method. DNase I activities were also detected by the DNase I activity staining after SDS–PAGE (Fig.1). The supernatant fraction from pETDNaseI-transformed cells, with or without IPTG induction (lanes 3 and 2, respectively), showed the same DNase I active band at the 29 kDa position, except that the band was much darker with induction, indicating that the production of rb-DNase I was greatly enhanced by IPTG. An endogenous E. coli DNase at the 20 kDa position was apparent in all the transformed cells, including cells transformed by the vector only (lane 1).
Fig. 1. Activity-stained gel of SDS–PAGE. The method of Lacks et al. (1979) was used for SDS–PAGE and the DNase I activity staining.The supernatant fractions of the vector only ( lane 1), the non-induced (lane 2) and induced ( lane 3) cells were applied. Each lane contained 15 mg of total protein. The purified bp-DNase I (0.3 units) was used in lane 4.[2]
References:
Colonies of E. coli strain BL21(DE3)pLysE transformed with pETDNaseI were cultured at 37°C in LB containing 50 mg of ampicillin/ml. IPTG induction (1mM) was doen when the absorbance of the culture medium (at 600 nm) reached approximately 1. After 3h of induction, centrifugation at 2000g (20min) was done to sediment the cells and cell debris. Due to the large amount of cell lysis after induction with IPTG, DNase I activities were found in both the cell pellet and supernatant fractions. The approximate yield from the induced culture was 3500 units/l as measured by the hyperchromicity assay method. DNase I activities were also detected by the DNase I activity staining after SDS–PAGE (Fig.1). The supernatant fraction from pETDNaseI-transformed cells, with or without IPTG induction (lanes 3 and 2, respectively), showed the same DNase I active band at the 29 kDa position, except that the band was much darker with induction, indicating that the production of rb-DNase I was greatly enhanced by IPTG. An endogenous E. coli DNase at the 20 kDa position was apparent in all the transformed cells, including cells transformed by the vector only (lane 1).
Fig. 1. Activity-stained gel of SDS–PAGE. The method of Lacks et al. (1979) was used for SDS–PAGE and the DNase I activity staining.The supernatant fractions of the vector only ( lane 1), the non-induced (lane 2) and induced ( lane 3) cells were applied. Each lane contained 15 mg of total protein. The purified bp-DNase I (0.3 units) was used in lane 4.[2]
References:
- Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical biochemistry, 162(1), 156–159. https://doi.org/10.1006/abio.1987.9999
- Chen, C. Y., Lu, S. C., & Liao, T. H. (1998). Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme. Gene, 206(2), 181–184. https://doi.org/10.1016/s0378-1119(97)00582-9
Sequence and Features
Assembly Compatibility:
- 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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Categories
Parameters
| None |