Difference between revisions of "Team:Dundee/fmodal"

Figure 11: β-galactosidase Assay Under Anaerobic Conditions A graph of the results from the anaerobic β-galactosidase assay aimed to characterize the interaction between the two separated parts of OBP2A. The graph seems to suggest that the two subunits of OBP2A may not be interacting in vivo. This can be inferred from the fact that the millers activity of the OBP2A sample lies lower than the negative controls from within the experiment. As a result of this information, one more assay will be prepared in which the two subunits will be switched with regards to the bacterial two hybrid vectors to eliminate any possibility of this being a factor effecting the interaction between the two subunits.

Figure 1: Amplification of LS LS was amplified using primers which would allow for subsequent cloning into the high expression vector pQE80-L. Lane 1 shows that the PCR has been successful since the band corresponds to the expected size of 2100 bp.

Figure 2: Amplification of LS LS was amplified from the IDT plasmid using primers to allow cloning of the gene into pSB1C3. The observed band corresponds to the expected size of LS, 2100 bp.

Figure 3: Overexpression Assays of LS 5 ml cultures of M15pREP4 E.coli was grown to OD₆₀₀=0.6 then induced with a range of IPTG concentrations. Samples were left to grow for a further 6 hours at 37^oC, at which point they were spun down and the pellet resuspended in 2x Laemmli buffer. The samples were boiled for 10 minutes and then loaded on to an SDS gel.

Figure 4: Characterisation of Lanosterol Synthase 5 ml cultures of M15pREP4 E.coli was grown to OD₆₀₀=0.6 then induced with a range of IPTG concentrations. Samples were left to grow for a further 6 hours at 37^oC, at which point they were spun down and the pellet resuspended in 2x Laemmli buffer. The samples were boiled for 10 minutes and then loaded on to an SDS gel. The proteins were transferred to a nitrocellulose membrane and probed with an anti-His antibody.

Figure 5: Chromatogram following Affinity Chromatography of LS Using FPLC, the protein sample was loaded onto a nickel column and eluted with an increasing concentration of imidazole (green line). The fractions corresponding to the peak were ran on an SDS gel.

Figure 6: SDS Gel of Peak Fractions 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer an loaded onto a SDS gel.

Figure 1: Alignment of BBa_K1058008. Sequences from top to bottom: sequencing result from forward primer; expected sequence; reverse complement of sequencing result from reverse primer; sequencing result from intermediate primer. Colour code: Green = Prefix/Suffix; Pink = Annealing site of intermediate primer; Yellow = Mismatch in one sequence; Red = In frame stop codons.

Figure 2: 1% agarose gel after PCR of BBa_K1058008 with primers specific to the promoter region, Pchr, and the repressor region, chrB. Left: Pchr (expected size 169bp); Middle: 1kb+ ladder, Right: chrB (expected size 939bp). Amplification of chrB was not successful. Pchr was further processed by restriction digest with EcoRI and PstI.

Figure 3: 1% agarose gel after PCR of BBa_K1058008 with primers specific to the repressor region, chrB. Left: 1.5 µl DMSO; Middle: 1kb+ ladder, Right: 2.5 µl DMSO. Expected size for each: 939bp. The annealing temperature for the PCR amplification was lowered to 50°C in order to increase the chances of accomodating a primer that is not 100% complementary.

Figure 4: 1% agarose gel after PCR of gfp with corresponding primers. Left: GFP (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of chrB, using primers to add a BamHI restriction site in front of it and a 6-His-Tag and a HindIII restriction site at the end. (expected size of chrB: 939bp). Since amplification of chrB was unsuccessful, it will be repeated.

Figure 5: 1% agarose gel after restriction digest of pSB1C3-Pchr, pSB1C3-chrB (s), and pSB1C3-chrB (w) with EcoRI and PstI for confirmation of size. Ladder: 1kb+. Top: 1-4: Pchr 1-4; chrB (s) 1-2. Bottom: chrB (s) 3-4; chrB (w) 1-4. Expected sizes: Pchr: 169bp; chrB: 939bp.

Figure 6: Alignment of Pchr sequence retrieved from BBa_K1058008 with the sequencing result of this region. Colour code: Green = Prefix/Suffix

Figure 7: Alignment of chrB (opt) sequence retrieved from BBa_K1058008 with the sequencing result of this region. Colour code: Green: Prefix and Suffix; Red: In frame stop codons; Blue: First 15 codons optimised for expression in E. coli.

Figure 8: 1% agarose gel after restriction digest of pUniprom (Expected size 2454bp).

Figure 11: 1% agarose gel after restriction digest of pSB1C3 with EcoRI and SpeI (Expected size 2070bp).

Figure 9: 1% agarose gel after PCR of chrB (opt). (Expected size 939bp).

Figure 10: Plates after transformation of Top: pUnipromchrB, Middle: pUnipromchrB (opt) and Bottom: pSB1C3-Pchr-gfp into JM110.

Figure 12: 1% agarose gel after restriction digest of pUnipromchrB, pUnipromchrB (opt) and pSB1C3-Pchr-gfp for confirmation of size. Ladder: 1kb+. Top: 1-4: pSB1C3-Pchr-gfp 1-4; pUnipromchrB (opt) 1-2. Bottom: pUnipromchrB (opt) 3-4; pUnipromchrB 1-4. Expected sizes: Pchr-gfp: 909bp; chrB and chrB (opt): 939bp.

Figure 13: 1% agarose gel after colony PCR of pUnipromchrB (opt). Top: colonies 1-11; Bottom: colonies 12-22. Colonies 2, 4, 9, and 12 were selected for sequencing. Ladder: 1kb+ Expected size of chrB (opt): 939bp.

Figure 14: Sequencing result of pSB1C3-Pchr-gfp after double ligation. Even though the sequence is as expected, it is ligated in reverse. It was hence called pSB1C3-Pchr-gfp (A) The Result for pSB1C3-Pchr-gfp (colony 2) was not as expected, albeit in the correct orientation. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.

Figure 15: Plates with pSB1C3-Pchr-gfp in JM110 after double ligation under UV light. Numbered colonies were selected for initial size confirmation and sequencing. However, other colonies seemed to express GFP as well, hence a second attempt was made with different colonies from the same plate.

Figure 16: Alignment of sequencing results of pSB1C3-Pchr-gfp - colonies 1, 5, 6, and 7 with sequences of gfp (mut2) and Pchr sequence. Colony 1 remained consistent, the other tested colonies were without success. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.

Figure 17: 1% Agarose gel after restriction digest of pUniprom with BamHI/PstI (expected size: 2454bp). Right: PCR product of Haptoglobin.

Figure 18: Plates after transformation of Pchr into pSB1C3. Top: ratio insert:vector 2:1; Bottom: ratio insert:vector 3:1.

Figure 19: Alignment of Pchr sequence retrieved from BBa_K1058008 with the sequencing result of this region. Colour code: Green = Prefix/Suffix

Figure 20: Plates after transformation of, Top: chrB into pUniprom; Bottom: chrB (opt) into pUniprom. Ratio insert:vector for ligations: left: 3:1; Right: 4:1.

Figure 21: Gel after restriction digest of Left: pUniprom-chrB, and Right: pUniprom-chrB (opt) with BamHI/PstI for confirmation of insert size. Expected size 939bp. chrB colony 2 and chrB (opt) colony 3 were selected for sequencing.

Figure 22: Alignments of: Left: chrB and Right: chrB (opt). Colours: Pink = Restriction sites (BamHI/PstI); Blue = 6-His Tag; Red = In-frame stop codons; Cyan = optimised codons.

Figure 23: Plates after ligation of gfp into pSB1C3-Pchr. Left: Ratio Insert:Vector 2:1; Right: Ratio Insert:Vector 3:1.

Figure 24: Gel for size confirmation of pSB1C3-Pchr-gfp (Top). Bottom: Left - Haptoglobin, Right - Lanosterol Synthase. pSB1C3-Pchr-gfp (3) and pSB1C3-Pchr-gfp (4) were submitted for sequencing.

Figure 25: Alignment of Pchr sequence retrieved from BBa_K1058008 and gfp (mut2) with the forward and reverse sequencing results of Pchr-gfp this region. The sequence was as expected, it was hence called pSB1C3-Pchr-gfp (B). Colour code: Green = Prefix/Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.

Figure 26: Top: anti-GFP: Single colonies of JM110 + pSB1C3-Pchr-gfp (A), MC1061 + pSB1C3-Pchr-gfp (B) were used to inoculate 5 ml of LB broth supplemented with 100 ^µg/_ml chloramphenicol. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 1 ml TBS. 100 µl of each sample was mixed with 100 µl laemmli buffer, and boiled for 10min. 3 µl of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and PmanA-gfp as a positive control. Bottom: anti-6-His - ChrB: Single colonies of JM110 pUniprom-chrB and JM110 pUniprom-chrB (opt) were used to inoculate 5 ml of LB broth supplemented with 100 ^µg/_ml ampicillin. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 200 µl laemmli buffer, and the sample boiled for 10min. 10 µl of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3 was included as a negative control, and hydA - 6-His as a positive control. Expected sizes: GFP ~ 27kDa, ChrB ~35kDa.

Figure 27: Plates after transformation of single constituents of chromate sensor into E. coli MG1655. From top to bottom: pUniprom-chrB (from colony 2 - 29/07), pUniprom-chrB (opt) (from colony 3 - 29/07), pSB1C3-Pchr-gfp (B), pSB1C3-Pchr-gfp (A), BBa_K1058008.

Figure 28: Western blot against GFP: Single colonies of MG1655, containing all 4 combinations, pUniprom-chrB - pSB1C3-Pchr-gfp (A), pUniprom-chrB - pSB1C3-Pchr-gfp (B), pUniprom-chrB (opt) - pSB1C3-Pchr-gfp (A), and pUniprom-chrB (opt) - pSB1C3-Pchr-gfp (B) were used to inoculate 5 ml of LB broth supplemented with 100 ^µg/_ml chloramphenicol and 100 ^µg/_ml ampicillin. After 16h of incubation at 37°C with agitation at 200rpm, each sample was subcultured into 5 ml of fresh, equally supplemented LB and cells were grown for 2 hours more. 1 ml of the subculture was then retrieved and pelleted. The pellet was resuspended in 1 ml PBS. 100 µl of these samples was mixed with 100 µl laemmli buffer, and the sample boiled for 10 min. The indicated volume of each sample was loaded on a SDS gel (12% acrylamide). pSB1C3, and pUniprom were included as a negative control, and PmanA-gfp as a positive control. The blot against 6-His – ChrB was without success and is not shown. No chromate of any kind was added to the medium previous to this blot, hence no expression of GFP was expected.

Figure 29: F,OD-plot of results of plate reader experiment after exposing MG1655 + BBa_K1058008 to a range of concentrations between 10 nM and 100 µM of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄. One measurement was made every 10min across 16h.

Figure 30: F,OD-plot of results of plate reader experiment after exposing MG1655 + pSB1C3-Pchr-gfp (B) + pUniprom-chrB to a range of concentrations between 10 nM and 100 µM of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄. One measurement was made every 10min across 16h.

Figure 31: F,OD-plot of results of plate reader experiment after exposing MG1655 + pSB1C3-Pchr-gfp (B) + pUniprom-chrB (opt) to a range of concentrations between 10 nM and 100 µM of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄. One measurement was made every 10min across 16h.

Figure 32: F,OD-plot of results of plate reader experiment after exposing MG1655 + pSB1C3-Pchr-gfp (B) to a range of concentrations between 10 nM and 100 µM of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄. One measurement was made every 10min across 16h.

Figure 33: Plates after ligation of chrB (Bielefeld) into pT7KS. From top to bottom: Ratio insert:vector 2:1; Ratio Insert:Vector 3:1; pT7KS religation control.

Figure 34: Plates after transformation of single constituents of chromate sensor into BL21 (DE3). From top to bottom: pSB1C3-Pchr-gfp (B) + pUniprom-chrB, pSB1C3-Pchr-gfp (B) + pUniprom-chrB (opt), pUniprom-chrB, and pUniprom-chrB (opt).

Figure 35: 1% agarose gel after colony PCR of pT7KS-chrB (Bielefeld). Top: colonies 1-6; Bottom: colonies 7-12. Ladder: 1kb+ Expected size of chrB (Bielefeld): 939bp

Figure 36: F,OD-plot of results of plate reader experiment after inducing expression of ChrB from the T7 promoter of pUniprom BL21 (DE3) pSB1C3-Pchr-gfp (B) + pUniprom-chrB, and pSB1C3-Pchr-gfp (B) + pUniprom-chrB (opt). IPTG was added after 4 hours. One measurement was made every 10min across 22h.