Table 1.
Composition of the trace element solution and the various combinations of terrific broth used in the experimental setup.
Figure 1.
Schematic Diagram of CYP1B1 gene.
The CYP1B1 gene has 2 introns and 3 exons. The open reading frame starts from Exon II and ends within Exon III. The gene codes for a 543 aminoacid protein with a membrane spanning domain at the N-terminal followed by a proline rich hinge region which in turn is followed by the cytosolic globular domain. The chromosomal location of the gene is 2p21–22.
Figure 2.
PAGE/Western blot pictures of CYP1B1 expression for various variables with absorption spectrum adjoining.
Each unit shows absorption spectrum of the expressed protein at 350 to 600 nm. Each absorption spectrum shows a peak somewhere around 410–420 nm which is characteristic of the heme present in the CYP1B1 protein. The colours in each graph has a defined sequence which correlates with the SDS-PAGE picture shown just underneath each absorption curve. In every graph, the dark red colour corresponds to the second lane of the gel picture underneath (lane next to marker which is the first lane starting from the left). The dark green corresponds to the third lane, dark blue to the fourth, cyan colour to the fifth, sixth is pink and seventh is the fluorescent green. (A) CYP1B1 expression with three different vectors viz. pET28a+, pCWori(+) and pTRC-His. (B) CYP1B1 expression with different media viz. LB-broth, tryptone based TB, peptone based TB and TB made with 1∶1 mixture of tryptone and peptone. (C) CYP1B1 expression at different time points viz. 12 h, 20 h, 24 h and 30 h (D) CYP1B1 expression with six different strains of E. coli viz. DH5α, JM109, C100, DE3, Codon Plus and Pril. (E)Western blot showing the increase in protein expression with increasing IPTG concentration. Starting from left is marker folowed by expression without IPTG induction and then folowed by 0.2 mM, 0.4 mM, 0.6 mM and 0.8 mM IPTG concentrations. (F) Expression pattern of CYP1B1 at four different temperatures viz. 18°C, 25°C, 30°C and 37°C. (G) CYP1B1 expression without and with TES. (H) CYP1B1 expression pattern with different concentrations of thiamine viz. 0.5 mM, 1.0 mM and 1.5 mM. (I) Expression pattern of CYP1B1 at different δ-ALA concentrations viz. 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM. (J) CYP1B1 expression with and without double colony selection. (K) Expression pattern of CYP1B1 at four different rpm levels viz. 150, 170, 200, 250. (L) Overall experiment layout with all the variables and results obtained.
Table 2.
Panoramic view of the experiments carried out with the numerical values of the variables and the results obtained.