Difference between revisions of "Team:Dundee/fmodal"

Figure 1: Amplification of LbpA The image shows the gel ran of the LbpA PCR product. The expected size of the fragment was 2832 bp which corresponds to the observed band on the gel.

Figure 2: LbpA PCR. Lane 1: PCR Product, Lane 2: 1kb Ladder and Marker. The image shows the gel ran of the LbpA PCR product. The expected size of the fragment was 2832 bp which corresponds to the observed band on the gel.

Figure 3: LbpA PCR for Cloning into pQE80-L. Lane 1: LbpA PCR Product, Lane 2: 1kb Ladder and Marker The image shows the gel ran of the LbpA PCR product. The expected size of the fragment was 2832 bp which corresponds to the observed band on the gel.

Figure 4: LbpA Culture Samples. Lane 1: Sample from uninduced culture. Lane 2: Sample from culture induced with 1mM IPTG. It seems that on comparison of the two cultures, that inducing expression of LbpA causes the cells to die given the significant reduction of protein levels visible on the gel.

Figure 5: OD₆₀₀ Readings from Uninduced Culture and Induced Cultures (0.5 mM, 1 mM and 2 mM IPTG). The table shows that after being induced with IPTG the cultures stop growing since their OD₆₀₀ readings are notably lower than that of the uninduced control.

Figure 6: Growth Curve of Uninduced and Induced Cultures (0.5 mM, 1 mM and 2 mM IPTG) The graph shows that on comparison with the uninduced control, the induced cultures stop growing when LbpA expression is induced. After ~7 hours, it is possible that the emergence of mutants causes the the OD to rise again or the cells have adapted to the production of the foreign protein.

Figure 7: Growth Curve of Uninduced and Induced Cultures (0.5 mM, 1 mM and 2 mM IPTG) The graph shows that on comparison with the uninduced control, after reaching an OD₆₀₀ of 0.6, the induced cultures stop growing when LbpA starts to be expressed. Similar to the previous growth curve assay, after ~7 hours, it is possible that the emergence of mutants causes the the OD to rise again or the cells have dealt with the production of the foreign protein.

Figure 8: SDS Gel of Samples Taken from Uninduced Control and 1 mM IPTG Induced Culture 6 hours After Induction For the induced culture, a faint band just above the 100 kDa marker is observable on the gel which corresponds to the expected size of LbpA.

Figure 1: Gel of SBP PCR Product for Cloning into pQE80-L. Lane 1: SBP PCR Product 1 Lane 2: 1kb Ladder and Marker Lane 3: SBP PCR Product 2 Both bands observable on the gel correspond to the expected size of SBP which is ~600 bp.

Figure 2: SDS Gel of Uninduced and 1 mM IPTG Induced Cultures of PotD and SBP. For PotD, it is clear from the gel that it is being successfully overexpressed since there is an intense band present at the 37 kDa marker which corresponds to the expected size of PotD in the induced culture sample. For SBP, there is no band present which suggests SBP is being overexpressed.

Figure 3: Characterisation of PotD. Single colonies of MC1061 E.coli containing pQE80-L encoding PotD were used to inoculate 5ml of fresh LB growth medium supplemented with ampicillin. Once cultures reached an OD₆₀₀ of 0.6, one was induced with 1 mM IPTG and one was left uninduced as a control. They were left to grow for a further three hours, after which a 1ml sample was taken and the cells pelleted. The cells were resuspended in 100µl of Laemmli buffer, 20µl of which was separated by SDS-PAGE and transferred to a nitrocellulose membrane and probed with an anti-His antibody.

Figure 4: SDS Gel of Uninduced and 0.5mM, 1mM and 2mM IPTG Induced Cultures of SBP. It appears that upon induction of SBP with any concentration of IPTG the cells are dying given that there is a significant drop in protein levels observable on the gel.

Figure 5: Growth Curve of Uninduced and Induced Cultures (0.5 mM, 1 mM and 2 mM IPTG) The graph shows that on comparison with the uninduced control, the induced cultures stop growing when SBP expression is induced. After ~7 hours, it is possible that the emergence of mutants causes the the OD to rise again or the cells have adapted to the production of the foreign protein.

Figure 6: Growth Curve of Uninduced and Induced Cultures (0.5 mM, 1 mM and 2 mM IPTG) The graph shows that on comparison with the uninduced control, after reaching an OD₆₀₀ of 0.6, the induced cultures stop growing when SBP starts to be expressed. Similar to the previous growth curve assay, after ~7 hours, it is possible that the emergence of mutants causes the the OD to rise again or the cells have adapted to the production of the foreign protein.

Figure 7: SDS-PAGE of Fractions Obtained After Size Exclusion Chromatography of PotD 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100 V. The two bands observable on the gel correspond to the expected size of PotD of 38 kDa. Western Blotting will confirm if these proteins are PotD.

Figure 8: Characterisation of PotD following Purification. 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V. It was then transferred to a nitrocellulose membrane and probed with an anti-His antibody.

Figure 9: Characterisation of SBP following Purification. 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V. It was then transferred to a nitrocellulose membrane and probed with an anti-His antibody.

Figure 1: pSB1C3-hHBA and pSB1C3-hHBB (respectively) Pre-Sequencing Digest pSB1C3-hHBA and pSB1C3-hHBB minipreps were digested with EcoRI and PstI to check for presence of each insert. The bands observable on the gel correspond to the expected sizes for the pSB1C3 vector and both inserts.

Figure 2: Amplification of hHBA and hHBB hHBA and hHBB were amplified from the pSB1c3-hHBA and pSB1c3-hHBB minipreps obtained yesterday using primers to allow cloning of the genes into pT25 and pUT18 respectively. Although the band is fainter in the case of hHBA, the gel indicates the PCR has been successful.

Figure 3: Amplification of hHBA hHBA was amplified from the pSB1C3-hHBA using primers to allow cloning of the genes into pT25. The gel indicates that the PCR has been successful.

Figure 4: Amplification of hHBA and hHBB hHBA and hHBB were amplified from the pSB1c3-hHBA and pSB1C3-hHBB minipreps using primers to allow cloning of the genes into pQE80-L. The gel indicates that the PCR has been successful.

Figure 5: Pre-Sequence Digest of pQE80-L-hHBA and pQE80-L-hHBB Both pQE80-L-hHBA and pQE80-L-hHBB minipreps were digested with BamHI and KpnI to check for the presence of the inserts. The gel indicates that both inserts have been successfully cloned into pQE80-L.

Overexpression of hHBB and hHBA. Single colonies of M15[pREP4] E.coli containing pQE80-L encoding hHBB and pQE80-L encoding hHBA were used to inoculate 5ml of fresh LB growth medium supplemented with ampicillin and kanamycin. Once cultures reached an OD₆₀₀ of 0.6, they were induced with a range of IPTG concentrations. They were left to grow for a further three hours, after which a 1 ml sample was taken and the cells pelleted. The cells were resuspended in 100 µl of 2x Laemmli buffer, 20 µl of which was separated by SDS-PAGE.

Figure 7: Characterisation of hHBB. Single colonies of M15[pREP4] E.coli containing pQE80-L encoding hHBB were used to inoculate 5 ml of fresh LB growth medium supplemented with ampicillin and kanamycin. Once cultures reached an OD₆₀₀ of 0.6, they were induced with a range of IPTG concentrations. They were left to grow for a further three hours, after which a 1 ml sample was taken and the cells pelleted. The cells were resuspended in 100 µl of Laemmli buffer, 20 µl of which was separated by SDS-PAGE. The cells were resuspended in 100 µl of Laemmli buffer, 20 µl of which was separated by SDS-PAGE and transferred to a nitrocellulose membrane and probed with an anti-His antibody.

Figure 8: Amplification of hHBN for Cloning into pQE80-L PCR product from amplification of hHBN from IDT plasmid using primers for cloninig into pQE80-L. The gel indicates hHBN has been amplified successfully since the observed band corresponds to the expected size of 1200 bp.

Figure 9: SDS-PAGE of Fractions Obtained After Size Exclusion Chromatography of hHBB 10 µl of each fraction was mixed with 10 µl of laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V. The four bands observable on the gel correspond to the expected size of PotD of 16 kDa. Western Blotting will confirm if these proteins are hHBB.

Figure 10: Characterisation of hHBB following Purification. 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V. It was then transferred to a nitrocellulose membrane and probed with an anti-His antibody.

Figure 11: Amplification of hHBN for Cloning into pT25 PCR product from amplification of hHBN from IDT plasmid using primers for cloninig into pT25. The gel indicates hHBN has been amplified successfully since the observed band corresponds to the expected size of 1200 bp.

Figure 14: Characterisation of hHBN following Purification. 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V. It was then transferred to a nitrocellulose membrane and probed with an anti-His antibody.

Figure 15: Amplification of hHBN hHBN was amplified using primers to allow for subsequent cloning into the two hybrid vector pT25. The band observable on the gel corresponds to the expected size of hHBN of 1200 bp.

Figure 15: Amplification of hHBA hHBA was amplified using primers to allow for subsequent cloning into the two hybrid vector pT25. The band observable on the gel corresponds to the expected size of hHBA of

Figure 16: Amplification of hHBN hHBN was amplified using primers to allow for subsequent cloning into the two hybrid vector pUT18. The band observable on the gel corresponds to the expected size of hHBN of 1200 bp.

Figure 1: Amplification of OBP2A for insertion into pSB1C3 vector . This is the PCR product from the amplification of OBP2A from the IDT plasmid using primers for cloning into pSB1C3. The gel indicates OBP2A has been amplified successfully since the observed band corresponds to the expected size of 500 bp. The concentration of the gel extracted OBP2A is 335.53 ng/ul.

Figure 2: A subsequent gel was run of the amplified OBP2A after it was first excised from the gel in Figure 1 This was to determine if the gel extraction was successful. The gel indicates that OBP2A has been extracted from the gel successfully, since the observed band corresponds to the expected size of 500 bp. This can now be restricted in preparation for ligation into pSB1C3.

Figure 3: A Gel Analysis of Restriction Digest from the 24/6. This gel analysis was done to determine if the reason for the failed transformation was an improperly digested OBP2A. This gel indicates that the restricted product hasn't been lost as the OBP2A gene fragment can be seen as a distinct band at 500 bp. A re-ligation of OBP2A into the pSB1C3 backbone will now occur.

Figure 4: Presequence Digest of pSB1C3-OBP2A. All seven plasmid purifications of the overnight cultures done on the 26/6 were digested to see if they contained the OBP2A insert. It is clear from the gel that all but sample 3 contain the insert for OBP2A as they all have faint bands at 500 bp's which corresponds to the size of OBP2A. It also appears that sample 5 contains the highest concentration, so that will be the sample that is sent for sequencing.

Figure 5: Presequence digest of pUT18-OBP2A To determine if any samples contain the OBP2A insert. It can be seen from this gel that of the 5 samples that were digested only two are show to have the OBP2A insert, therefore sample 2 will be sent for sequencing.

Figure 6: Amplification of OBP2A for insertion into pT25 vector . This is the PCR product from the amplification of OBP2A for cloning into pT25. The gel indicates OBP2A has been amplified successfully since the observed band corresponds to the expected size of 100 bp.

Figure 7: Presequence Digest of pT25-OBP2A In order to determine if any of the samples contain the OBP2A insert. It can be seen from this gel that samples 5, 6 and 7 may have inserts in them as they each have two distinct bands, one of which corresponds to the 100 bp size for the OBP2A for pT25. As a result all three samples will be sent for sequencing.

Figure 8: β-galactosidase Assay A graph of the results from the first attempt at a β-galactosidase assay that aimed to characterize the interaction between the two separated parts of OBP2A. As it can be seen that the positive result has failed(far left) not much can be inferred from the interaction of the two parts of OBP2A. However it looks like the two subunits of OBP2A may not be interacting. As this was a first attempt, subsequent assays will be performed in order to determine if in fact the two separated parts of OBP2A aren't interacting in vivo - like these results suggests.

Figure 9: β-galactosidase Assay A graph of the results from the second attempt at a β-galactosidase assay that aimed to characterize the interaction between the two separated parts of OBP2A. It can also be inferred from this graph that the two parts of OBP2A don't seem to be interacting as it has a low millers activity when compared to the controls. Further assays will be performed in order to determine if the two separated parts of OBP2A aren't interacting in vivo like these results suggests.