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Revision as of 19:03, 17 September 2015
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Summary
Lactoferrin binding protein A (LbpA) was successfully cloned into the vector pSB1C3.
Aim of experiment: To digest LbpA with BamHI and EcoRI and then ligate it into pSB1C3.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps: Ligations of pSB1C3-LbpA were left overnight until the next day when they would be transformed into the JM110 E.coli.
Aim of experiment: To transform the pSB1C3-LbpA ligations into JM110 E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: Transformations will be checked tomorrow and if successful, overnight cultures will be set up.
Aim of experiment: To set up overnight cultures of JM110 E.coli containing pSB1C3-LbpA.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: A miniprep will be performed tomorrow on the overnight cultures.
Aim of experiment: To miniprep the JM110 E.coli overnight cultures containing pSB1C3-LbpA.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: The miniprep with the highest concentration will be sent for sequencing.
Summary
Sequencing confirmed that LbpA had been successfully cloned into pSB1C3 therefore this week cloning was attempted to put LbpA into the high expression vector pQE80-L.
Aim of experiment: To amplify LbpA for subsequent cloninng into the pQE80-L vector.
Protocols Used: PCR
Results: N/A
Next Steps: The PCR product will be digested and ligated into pQE80-L tomorrrow.
Aim of experiment: To restrict and ligate the LbpA gene into the pQE80-L plasmid.
Protocols Used: Restriction Digests Ligations
Results:: N/A
Next Steps: Transformations of pQE80-L-LbpA into JM110 E.coli will be carried out tomorrow.
Aim of experiment: To transform pQE80-L-LpbA into JM110 E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: If the transformations are successful, colonies will be picked to set up overnight cultures.
Summary
This week, cloning of LbpA into pQE80-L was continued due to last week's failed attempts.
Summary
LbpA was successfully cloned into the high expression vector pQE80-L.
Aim of experiment: To digest the amplified LbpA gene with BamHI and KpnI and subsequently ligate it into the pQE80-L vector.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps: The ligations will be transformed into M15[pREP4] E.coli.
Aim of experiment: To transform pQE80-L-LbpA into M15[pREP4] E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: Overnight cultures will be set up tomorrow if the transformations are successful.
Aim of experiment: To set up overnight cultures of M15[pREP4] E.coli containing pQE80-L-LbpA.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Overnight cultures will be miniprepped and a pre-sequence restriction digest will be performed to check for the presence of the LbpA gene.
Aim of experiment: To miniprep the overnight cultures from yesterday and perform a pre-sequencing restriction digest using BamHI and KpnI to check for presence of LbpA gene.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: A sample of the miniprep was sent for sequencing to confirm the presence of LbpA in the pQE80-L vector.
Summary
Protein expression optimization experiments were performed on LbpA to assay protein production levels. However, cell growth seems to cease when LbpA expression is induced.
Aim of experiment: To set up overnight cultures in preparation for protein expression experiments tomorrow.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be used to set up day cultures tomorrow which will be used to test expression of LbpA.
Aim of experiment: To set up day cultures and induce expression of LbpA using different concentrations of IPTG and check levels of protein production via SDS-PAGE.
Protocols Used: Protein Expression Optimization
Results: N/A
Next Steps: The samples were stored in the -20 oC freezer overnight and will be ran on a gel tomorrow.
Aim of experiment: To test the samples obtained yesterday on an SDS gel.
Protocols Used: Protein Expression Optimization
Results: Figure 4
Next Steps: Further optimization experiments will be set up next week using different concentrations of IPTG.
Summary
Further protein expression optimization experiments were set up, this time using a range of IPTG concentrations to induce expression of LbpA.
Aim of experiment: To set up further tests to optimize expression of LbpA using different concentrations of IPTG.
Protocols Used: Protein Expression Optimization Note: An SDS gel was not ran, instead the OD600 readings were recorded for the uninduced and induced cultures at 20 minute intervals for 2 hours, 1 and half hours after the cultures were induced.
Results: Figure 5
Next Steps: A plate reader experiment will be set up next week to monitor the growth rate of the the cells over a longer period of time after LbpA expression has been induced.
Summary
Protein expression optimization experiments were continued this week. A growth curve assay was carried out to assess the effect of LbpA expression over a longer period of time.
Aim of experiment: To induce expression of LbpA using different concentration of IPTG and allow cells to grow for 16 hours in order to monitor cell growth.
Protocols Used: Growth Curve Assay
Results: Figure 6
Next Steps: A plate reader experiment will be set up next week to monitor the growth rate of the the cells over a longer period of time after LbpA expression has been induced.
Aim of experiment: To check if the cells in the induced cultures ~7 hours after being induced are producing LbpA.
Protocols Used: Western Blot Sample Preparation Note: The day cultures were grown for 7 hours after they were induced and then the samples were frozen, to be blotted on Monday.
Results: N/A
Next Steps: The samples will be ran on an SDS gel on Monday and blotted.
Summary
Samples from the growth curve assay were blotted to see if LbpA is being produced at the later stages of the growth curve. A further growth curve assay was also performed which monitored cell growth until a certain OD600 was reached, at which point the cultures were induced.
Aim of experiment: To western blot the LbpA culture samples from Friday.
Protocols Used: Western Blot
Results: Nothing was visible on the blot.
Next Steps: Another plate reader experiment will be set up on Thursday where the cultures will be monitored for three hours before induction with IPTG and then for a further 20 hours afterwards.
Aim of experiment: To check if the cells begin to die after induction with IPTG after having already grown to an OD600 of 0.5.
Protocols Used: Growth Curve Assay
Results: Figure 7
Next Steps: Samples of these cultures will be taken 6 hours after induction to check for the presence of LbpA.
Summary
The presence of LbpA was confirmed in the culture 6 hours after induction with IPTG so protein purification was started this week.
Aim of experiment: To set up a 150 ml overnight culture for sub-culturing tomorrow.
Protocols Used: Overnight Cultures Note: 150 ml of LB was used and 150 µl of each antibiotic.
Results: N/A
Next Steps: 150 ml of fresh LB will be inoculated tomorrow with 7.5 ml of the overnight cultures.
Aim of experiment: To set up day cultures using the overnight cultures from yesterday and test for expression of LbpA 6 hours after induction.
Protocols Used: Protein Expression Test Note: 150 ml of fresh LB was inoculated with 7.5 ml of the overnight cultures. Only an uninduced control and an 1 mM IPTG induced culture was set up. The samples were not blotted, they were only ran on an SDS gel.
Results: Figure 8
Next Steps: A 3 L culture will be set up on Thursday under the same conditions as the overnight cultures in order to try and purify LbpA.
Aim of experiment: To set up overnight cultures for the 3L day cultures that will be grown tomorrow.
Protocols Used: Overnight Cultures Note: One 150 ml overnight culture was set up using 150 µl of each antibiotic.
Results: N/A
Next Steps: The 3 L culture will be set up tomorrow allowed to grow to OD600 of 0.6-1 and then induced with 1 mM IPTG and grown for 6 hours.
Aim of experiment: To set up 3 L day cultures for purifying LbpA.
Protocols Used: Protein Purification Note: The procedure was stopped at Step 6 and the pellets were frozen at -20 oC.
Results: N/A
Next Steps: Protein purification will be continued on Monday.
Summary
The first round of LbpA purification failed at the affinity chromatography stage so another 6 L culture was grown this week to attempt purification again.
Aim of experiment: To continue with purification of LbpA.
Protocols Used: Protein Purification Note: The protocol was continued from Step 7
Results: N/A
Next Steps:Size exclusion chromatography was not carried out since an SDS gel showed LbpA was not present after performing affinity chromatography. Another 6L culture will be set up this week.
Aim of experiment: To set up overnight cultures for the 6 L culture that will be set up tomorrow.
Protocols Used: Overnight Cultures Note: One 150 ml overnight culture was set up using 150 µl of each antibiotic.
Results: N/A
Next Steps:The overnight cultures will be used to set up the 6 L day culture tomorrow.
Aim of experiment: To set up 6 L day cultures for purifying LbpA and begin the purification process.
Protocols Used: Protein Purification Note: The protocol was stopped at Step 6.
Results: N/A
Next Steps: Purification of LbpA will be continued on Monday.
Summary
Purification of LbpA was continued but unfortunately was unsuccessful. If time permits, another round of purification may be reattempted.
Aim of experiment: To continue with purification of LbpA.
Protocols Used: Protein Purification Note: The protocol was continued from Step 7.
Results: N/A
Next Steps: Purification of LbpA will be continued tomorrow.
Aim of experiment: To purify LbpA using affinity chromatography.
Protocols Used: Protein Purification Note: The protocol was stopped at Step 13.
Results: N/A
Next Steps:Since no peaks were observed, purification of LbpA was abadoned at this stage. Purification may be retried.
Summary
Cloning of PotD into the biobrick vector, pSB1C3, was started this week.
Aim of Experiment: To amplify PotD from MG1655 E.coli gDNA.
Protocols Used: PCR
Results: N/A
Next Steps: The PCR product will be gel extracted, digested and ligated into the biobrick vector pSB1C3 tomorrow.
Aim of experiment: To gel extract the PotD PCR product and digest it with PstI and EcoRI. A ligation will then be set up with pSB1C3.
Protocols Used: Restriction Digest Ligation
Results: N/A
Next Steps: Ligations will be transformed into JM110 E.coli on Monday.
Summary
Cloning of PotD into the biobrick vector was completed this week.
Aim of experiment: To transform pSB1C3-PotD into JM110 E.coli.
Protocols Used: Transformations
Results:N/A
Next Steps: If the transformations are successful, overnight cultures will be set up tomorrow.
Aim of experiment: To set up overnight cultures of pSB1C3-PotD in JM110 E.coli
Protocols Used: Overnight Cultures
Results:: N/A
Next Steps: The overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep the overnight cultures from yesterday.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: The miniprep with the highest concentration will be sent for sequencing to confirm PotD has been inserted into pSB1C3 successfully.
Summary
This week, cloning of PotD into pQE80-L was started. The gene for Spermine Binding Protein (SBP) also arrived in a plasmid so it was transformed into MC1061 E.coli and plasmid purified.
Aim of experiment: To digest the PotD PCR product with KpnI and BamHI and then ligate it into pQE80-L. To transform MC1061 E.coli with the IDT plasmid containing SBP.
Protocols Used:
PotD: Restriction Digests Ligations SBP:TransformationsResults: N/A
Next Steps: The pQE80-L-PotD ligations will be transformed into MC1061 E.coli. Overnight cultures of the transformations will be set up tomorrow.
Aim of experiment: To transform the pQE80-L-PotD ligations into MC1061 E.coli. To set up overnight cultures of the transformations of MC1061 E.coli with SBP + IDT Plasmid that were set up yesterday.
Protocols Used: PotD: Transformations SBP: Overnight Cultures
Results:N/A
Next Steps: If transformations are successful, overnight cultures will be set up tomorrow.
Aim of experiment: To repeat ligations of PotD into pQE80-L since transformations did not work. To miniprep the overnight cultures that were set up yesterday.
Protocols Used: PotD: Ligations SBP: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: Ligations will be left over the weekend and transformed on Monday.
Summary
Cloning of PotD into the high expression vector pQE80-L was completed. Cloning of SBP into pSB1C3 and pQE80-L was started this week.
Aim of Experiment: To transform pQE80-L-PotD into MC1061E.coli. To set up a PCR of SBP using the purified plasmid from Friday and to gel extract the PCR product for cloning into pQE80-L. To digest SBP out of the same plasmid using EcoRI and PstI and subsequently ligate it into pSB1C3.
Protocols Used: PotD: Transformations SBP: PCR Restriction Digests Ligations
Results: SBP : Figure 1
Next Steps: Overnight cultures will be set up tomorrow for the PotD transformations. The SBP PCR product will be digested and ligated into pQE80-L tomorrow. The pSB1C3-SBP ligations will be transformed into MC1061 E.coli.
Aim of experiment: To set up overnight cultures of MC1061 E.coli containing pSB1C3 + PotD. To transform MC1061 E.coli with pSB1C3 + SBP. To digest the SBP PCR product with BamHI and Kpn I, ligate it into pQE80-L and transform these ligations into MC1061 E.coli.
Protocols Used: PotD: Overnight Cultures SBP: Transformations Restriction Digests Ligations
Results: N/A
Next Steps: The MC1061 E.coli overnight cultures containing pQE80-L + PotD will be miniiprepped tomorrow. Overnight cultures will be set up of the MC1061 E.coli transformations with pSB1C3 + SBP and pQE80-L + SBP.
Aim of experiment: To miniprep the overnight cultures of MC1061 E.coli containing pSB1C3-PotD. To set up overnight cultures of the transformations of MC1061 E.coli with pSB1C3-SBP and pQE80-L-SBP
Protocols Used: PotD: Plasmid Purification (QIAprep® Spin Miniprep Kit) SBP: Overnight Cultures
Results: N/A
Next Steps: The miniprep with the highest concentration for pQE80-L + PotD was sent for sequencing. The SBP overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep the overnight cultures of MC1061 E.coli containing pSB1C3-SBP and pQE80-L + SBP.
Protocols Used: SBP: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: The miniprep with the highest concentration for both pSB1C3-SBP and pQE80-L-SBP were sent for sequencing.
Summary
Sequencing showed SBP failed to insert successfully into pSB1C3 so cloning was repeated from the ligation stage this week. However, SBP and PotD were successfully inserted into the high expression vector pQE80-L, so overexpression assays were started this week for both.
Aim of Experiment: To ligate SBP into pSB1C3 and transform the ligations into MC1061 E.coli.
Protocols Used: SBP: Ligations Transformations
Results: SBP : Figure 1
Next Steps: Overnight cultures will be set up tomorrow if transformations are successful.
Aim of experiment: To set up overnight cultures of MC1061 E.coli containing pSB1C3-SBP. To set up overnight cultures of pQE80-L-SBP and pQE80-L + PotD in MC1061 E.coli for overexpression assays to be performed tomorrow.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The MC1061 E.coli overnight cultures containing pSB1C3- SBP will be miniiprepped tomorrow. The MC1061 E.coli overnight cultures containing pQE80-L- SBP and pQE80-L-PotD will be subcultured tomorrow in order to perform overexpression assays.
Aim of experiment: To miniprep the overnight cultures of MC1061 E.coli containing pSB1C3-SBP. To start protein expression optimization for PotD and SBP.
Protocols Used: SBP: Plasmid Purification (QIAprep® Spin Miniprep Kit) SBP and PotD: Protein Expression Optimization Note: Protocol was stopped at Step 8. Samples will be ran on an SDS gel tomorrow.
Results: N/A
Next Steps: The miniprep with the highest concentration for pSB1C3 + SBP was sent for sequencing. The PotD and SBP samples will be ran on an SDS gel tomorrow.
Aim of experiment: To run the samples of PotD and SBP on an SDS gel and begin Western Blotting.
Protocols Used: PotD and SBP: Protein Expression Optimization Note: Protocol was continued from Step 9.
Results: Figure 2
Next Steps: The membrane for the Western blot has been left at the blocking stage so will be continued tomorrow.
Aim of experiment: To continue the Western Blot of PotD and SBP.
Protocols Used: PotD and SBP: Western Blot
Results: Figure 3
Next Steps: PotD has been successfully characterized so purification of this protein will be started next week. Further expression optimization experiments for SBP will be set up next week using different concentrations of IPTG, since its characterization was unsuccessful.
Summary
Further protein expression optimization experiments were set up, this time using a range of IPTG concentrations to induce expression of SBP.
Aim of experiment: To set up further tests to optimize expression of SBP using different concentrations of IPTG.
Protocols Used: Protein Expression Optimization Note: Cultures were induced with 0.5 mM, 1 mM and 2 mM IPTG. The Western Blot was left to block overnight.
Results: Figure 4
Next Steps: The Western Blot will be completed tomorrow.
Aim of experiment: To complete the Western Blot that was started yesterday.
Protocols Used: Western Blot Note: Protocol was continued from Step 12.
Results: Nothing was visible on the blot.
Next Steps: A plate reader experiment will be set up next week to monitor the growth rate of the the cells over a longer period of time after SBP expression has been induced.
Summary
Protein expression optimization experiments were continued this week. A growth curve assay was carried out to assess the effect of SBP expression over a longer period of time. Purification of PotD was also started this week.
Aim of experiment: To induce expression of SBP using different concentration of IPTG and allow cells to grow for 16 hours in order to monitor cell growth.
Protocols Used: Growth Curve Assay
Results: Figure 5
Next Steps: A plate reader experiment will be set up next week to monitor the growth rate of the the cells over a longer period of time after SBP expression has been induced.
Aim of experiment: To set up overnight cultures that will be used tomorrow to set up 3 L cultures for PotD purification.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The 3 L day culture will be set up tomorrow.
Aim of experiment: To check if the cells in the induced cultures ~7 hours after being induced are producing SBP. To set up 3 L Day Culture for PotD Purification
Protocols Used: SBP: Western Blot Sample Preparation Note: The day cultures were grown for 7 hours after they were induced and then the samples were frozen, to be blotted on Monday. PotD: Protein Purification Note: Cultures were left to grow overnight after being induced.
Results: N/A
Next Steps: The samples will be ran on an SDS gel on Monday and blotted. The PotD cultures will be harvested tomorrow.
Aim of experiment: To harvest the day cultures grown yesterday for PotD purification.
Protocols Used: Protein Purification Note: After the cultures were spun down the pellets were frozen.
Results: N/A
Next Steps: Purification of PotD will continue on Monday.
Summary
Samples from the growth curve assay were blotted to see if SBP is being produced at the later stages of the growth curve. A further growth curve assay was also performed which monitored cell growth until a certain OD600 was reached, at which point the cultures were induced. Purification of PotD was also continued this week.
Aim of experiment: To western blot the SBP culture samples from Friday. To continue purfying PotD.
Protocols Used: SBP: Western Blot PotD: Protein Purification Note: Affinity chromatography was completed but no peaks were observable.
Results: Nothing was visible on the blot.
Next Steps: Another plate reader experiment will be set up on Thursday where the cultures will be monitored for three hours before induction with IPTG and then for a further 20 hours afterwards. Another 150 ml overnight culture was set up for PotD so 3 L cultures could be set up tomorrow.
Aim of experiment: To set up 3 L Day Culture for PotD Purification.
Protocols Used: Protein Purification Note: Cultures were left to grow overnight after being induced.
Results: N/A
Next Steps: Purification will be continued tomorrow.
Aim of experiment: To continue purification of PotD.
Protocols Used: Protein Purification Note: The protocol was continued from Step 4 and stopped at Step 13.
Results: N/A
Next Steps: Purification will be continued tomorrow.
Aim of experiment: To continue purification of PotD and to check if the cells begin to die after inducing expression of SBP with IPTG after having already grown to an OD600 of 0.5.
Protocols Used: PotD: Protein Purification Note: The protocol was continued from Step 13. SBP: Growth Curve Assay
Results: Figure 6
Next Steps: The samples from the size exclusion chromatography performed on PotD will be prepared tomorrow for blotting next week. For SBP further overexpression tests will be set up next week.
Summary
PotD was successfully purified and characterized this week. SBP was still unobservable in induced 150ml cultures so 3 L cultures containing pQE80-L-SBP were set up.
Aim of experiment: To set up a 150 ml overnight culture containing pQE80-L-SBP for sub-culturing tomorrow and to run an SDS-PAGE and Western Blot the PotD samples from Friday.
Protocols Used: SBP: Overnight Cultures Note: 150 ml of LB was used and 150 µl of each antibiotic. PotD: SDS-PAGE and Western Blotting Note: The membrane was left blocking overnight.
Results: N/A
Next Steps: 150 ml of fresh LB will be inoculated tomorrow with 7.5 ml of the overnight cultures containing pQE80-L-SBP. The PotD Western Blot will be completed tomorrow.
Aim of experiment: To set up day cultures using the overnight cultures from yesterday and test for expression of LbpA 6 hours after induction.
Protocols Used: Protein Expression Test Note: 150 ml of fresh LB was innoculated with 7.5ml of the overnight cultures. Only an uninduced control and an 1 mM IPTG induced culture was set up. The samples were not blotted, they were only ran on an SDS gel.
Results: SBP: Nothing was observable on the SDS gel. PotD: Figure 8
Next Steps: Since PotD has now been successful purified, the next step is to attach a fluorescent nanobead to the protein. Hopefully, this will be attempted in the coming weeks. Even though SBP was not visible on the SDS gel, 3 L cultures will be set up this week to try and harvest the protein since the culture size may be too small.
Aim of experiment: To set up overnight cultures containing pQE80-L-SBP for the 3L day cultures that will be grown tomorrow.
Protocols Used: Overnight Cultures Note: One 150ml overnight culture was set up using 150µl of each antibiotic.
Results: N/A
Next Steps: The 3L culture will be set up tomorrow allowed to grow to OD600 of 0.6-1 and then SBP expression induced with 1mM IPTG and grown for 6 hours.
Aim of experiment: To set up 3 L day cultures for purifying SBP.
Protocols Used: Protein Purification Note: The procedure was stopped at Step 6 and the pellets were frozen at -20oC.
Results: N/A
Next Steps: Protein purification will be continued on Monday.
Summary
It seems that SBP has been successfully purified and characterized. However, there was still a lot of contamination in the samples after size eclusion chromatography was performed. .
Aim of experiment: To continue with purification of SBP.
Protocols Used: Protein Purification Note: The protocol was continued from Step 7
Results: N/A
Next Steps: Size exclusion chromatography will be carried out tomorrow.
Aim of experiment: To continue purification of SBP.
Protocols Used: Protein Purification Note: The protocol was continued from Step 14.
Results: N/A
Next Steps: The fractions corresponding to the peak observed during SEC, will be ran on an SDS gel and Western Blotted tomorrow.
Aim of experiment: To perform a Western Blot on the samples obtained from Size Exclusion Chromatography yesterday..
Protocols Used: Western Blot
Results: Figure 9
Next Steps: There was still some contamination in the gel filtrated samples so this will have to be removed before a nanobead can be attached to the protein.
Summary
Interactions between PotD and spermidine were analysed via tryptophan fluorescence this week.
Aim of experiment: To analyse the interaction of PotD and spermidine using tryptophan fluorescence.
Protocols Used: Tryptophan Fluorescence
Results: N/A
Next Steps:
Summary
Cloning of hHBA and hHBB into the biobrick vector, pSB1C3, was performed this week.
Aim of Experiment: To digest hHBA and hHBB from IDT gBlocks using EcoRI and PstI for ligation into pSB1C3.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps: The pSB1C3-hHBA and pSB1C3-hHBB ligations will be transformed into JM110 E.coli.
Aim of experiment: To transform the pSB1C3-hHBA and pSB1C3-hHBB ligations into JM1110 E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: If transformations are successful, overnight cultures will be set up.
Summary
The sequence for pSB1C3-hHBB was confirmed this week so cloning of hHBB into the two hybrid system vector pUT18 was started. Sequencing of pSB1C3-hHBA was incorrect so another miniprep was sent for sequencing.
Aim of experiment: To set up overnight cultures of JM110 E.coli containing pSB1C3-hHBA and pSB1C3-hHBB from the transformations performed on Tuesday.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Overnight cultures will be miniprepped tomorrow and a test restriction digest will be performed to check for the presence of the inserts.
Aim of experiment: To miniprep the JM110 E.coli overnight cultures containing pSB1C3-hHBA and pSB1C3-hHBB and perform a pre-sequence digest check.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit) Restriction Digests
Results: Figure 1
Next Steps: The gel indicates that hHBA and hHBB have inserted successfully into pSB1C3 so samples of the minipreps will be sent for sequencing to confirm this.
Aim of experiment: To amplify hHBA and hHBB for Cloning into the Two Hybrid System Vectors pT25 and pUT18, respectively.
Protocols Used: PCR
Results: Figure 2
Next Steps: Sequencing of pSB1C3-hHBA and pSB1C3-hHBB showed that pSB1c3-hHBA had a 1 bp deletion so one of the other minipreps was sent for sequencing. pSB1C3-hHBB, however, was successful so the band for hHBB will be gel extracted and digested tomorrow.
Aim of experiment: To gel extract and digest hHBB with BamHI and EcoRI.
Protocols Used: Restriction Digests
Results: N/A
Next Steps: Ligations of hHBB will be set up tomorrow.
Aim of experiment: To set up ligations of pUT18-hHBB.
Protocols Used: Ligations
Results: N/A
Next Steps: Ligations will be left over the weekend and transformed into BTH101 E.coli.
Summary
Cloning of hHBA and hHBB into pT25 and pUT18, respectively, was continued this week.
Aim of experiment: To transform pUT18-hHBB into BTH101 E.coli.
Protocols Used: Transformations
Results:N/A
Next Steps: If transformations are successful, overnight cultures will be set up tomorrow. Sequencing showed the sequence for pSB1C3-hHBA was incorrect again, a third miniprep will be sent for sequencing.
Aim of experiment: To set up overnight cultures of BTH101 E.coli containing pUT18-hHBB from the transformations carried out yesterday.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be miniprepped and sent for sequencing tomorrow.
Aim of experiment: To miniprep the BTH101 E.coli Overnight Cultures containing pUT18-hHBB.
Protocols Used:
Plasmid Purification (QIAprep® Spin Miniprep Kit)Results: N/A
Next Steps: The miniprep with the highest concentration will be sent for sequencing to confirm presence of insert. Sequencing for pSB1C3-hHBA came back correct so another PCR of hHBA will be set up tomorrow for cloning into pT25.
Aim of experiment: To digest the hHBA PCR product with BamHI and KpnI and set up ligations with pT25.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps: Ligations will be left over the weekend and transformed into BTH101 E.coli on Monday.
Summary
Cloning of hHBA into pT25 was completed this week. Sequencing confirmed that hHBB has been successfully inserted into pUT18. Cloning of both hHBA and hHBB, individually, into the high expression vector pQE80-L was started this week.
Aim of Experiment: To transform pT25-hHBA into BTH101 E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: Overnight cultures will be set up tomorrow for the pT25-hHBA transformations if successful. Sequencing confirmed hHBB has been successfully inserted into pUT18.
Aim of experiment: To set up overnight cultures of MC1061 E.coli containing pT25-hHBA. To amplify hHBA.and hHBB for cloning into pQE80-L.
Protocols Used: Overnight Cultures Transformations Restriction Digests Ligations
Results: Figure 4
Next Steps: The BTH101 E.coli overnight cultures containing pT25-hHBB will be miniiprepped tomorrow. The PCR products will be digested and ligate dinto pQE80-L tomorrow.
Aim of experiment: To miniprep the overnight cultures of BTH101 E.coli containing pT25-hHBA. To digest hHBA and hHBB with BamHI and KpnI and set up ligations with pQE80-L.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit) Restriction Digests Ligations
Results: N/A
Next Steps: The miniprep with the highest concentration for pT25-hHBA will be sent for sequencing. The pQE80-L-hHBA and pQE80-L-hHBB will be transformed into M15[pREP4] E.coli tomrorrow.
Aim of experiment: To transform the pQE80-L-hHBA and pQE80-L-hHBB ligations into M15[pREP4] E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: If transformations are successful, overnight cultures will be set up on Monday.
Summary
Cloning of hHBA and hHBB into pQE80-L was completed this week. Cloning of Haptoglobin (hHBN) was also started this week.
Aim of Experiment: To set up overnight cultures of BTH101 E.coli containing pQE80-L-hHBA and pQE80-L-hHBB.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps:The overnight cultures will be miniprepped tomorrow. Sequencing for pT25-hHBA was incorrect so cloning will be repeated from the restriction digest stage.
Aim of experiment: To set up miniprep the BTH101 E.coli Overnight Cultures containing pQE80-L-hHBA and pQE80-L-hHBB.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: Figure 5
Next Steps: The pre-sequence digest indicates that both hHBA and hHBB have been inserted into pQE80-L successfully. The minipreps with the highest concentration will be sent for sequencing. The sequence for Haptoglobin (hHBN) arrived in an IDT plasmid, work will be be started on this tomorrow.
Aim of experiment: To transform the IDT Plasmid-hHBN into MC1061 E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: Overnight cultures will be set up tomorrow.
Aim of experiment: To set up overnight cultures of MC1061 E.coli containing IDT Plasmid-hHBN.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be miniprepped tomorrow and hHBN will be amplified for cloning into pSB1C3.
Aim of experiment: To miniprep the MC1061 E.coli containing IDT Plasmid-hHBN and to digest hHBN out of the plasmid using EcoRI and PstI.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit) Restriction Digests
Results: N/A
Next Steps: hHBN will be ligated into pSB1C3 on Monday. Sequencing also confirmed hHBA and hHBB have been successfully inserted into pQE80-L.
Summary
hHBB was successfully characterized this week so protein purification was started. Cloning of hHBN into pSB1C3 was also completed.
Aim of experiment: To ligate hHBN into pSB1C3.
Protocols Used: Ligations Overnight Cultures
Results:N/A
Next Steps: The pSB1C3-hHBN ligations will be transformed into MC1061 E.coli tomorrow. The overnight cultures will be subcultured tomorrow and induced with IPTG to test protein expression levels.
Aim of experiment: To transform pSB1C3-hHBN into MC1061 E.coli and set up expression optimization tests for hHBA and hHBB.
Protocols Used: Transformations Protein Expression Optimization
Results: Figure 6
Next Steps: If transformations are successful, overnight cultures will be set up tomorrow. Western Blotting will be continued tomorrow for hHBA and hHBB.
Aim of experiment: To set up overnight culture of MC1061 E.coli containing pSB1C3-hHBN and finish Western Blotting of hHBA and hHBB.
Protocols Used: Overnight Cultures Western Blotting
Results: Figure 7
Next Steps: The blot shows that characterization of hHBB has been successful. Unfortunately, nothing was visible on the blot for hHBA. 3 L cultures for purification of hHBB will be set up this week. Characterization of hHBA may be reattempted. The overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep the MC1061 E.coli overnight cultures containing pSB1C3-hHBN and set up overnight cultures of M15[pREP4] E.coli containing pQE80-L-hHBB for 3L cultures.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit) Overnight Cultures
Results: N/A
Next Steps: The miniprep with the highest concentration will be sent for sequencing. The overnight cultures will be used to set up 3L day cultures tomorrow for purrification of hHBB.
Aim of experiment: To set up 3 L day cultures of M15[pREP4] E.coli containing pQE80-L-hHBB for purification of hHBB.
Protocols Used: Protein Purification Note: The protocol was stopped at Step 6.
Results: N/A
Next Steps: Purification will be continued on Monday.
Summary
hHBB was successfully purified this week and cloning of hHBN into pQE80-L was completed.
Aim of experiment: To amplify hHBN for cloning into pQE80-L and continuation of hHBB purification.
Protocols Used: PCR Protein Purification Note: Protocol was continued from Step 6.
Results: Figure 8
Next Steps: The fractions from the affinity chromatography will be consolidated and concentrated tomorrow. Size exclusion chromatography will also be performed. The hHBN PCR product will be digested and ligated into pQE80-L tomorrow.
Aim of experiment: To consolidate, concentrate and perform size exclusion chromatography on the hHBB samples. To digest the hHBN PCR product with BamHI and KpnI and subsequently ligate it into pQE80-L.
Protocols Used: Protein Purification SDS Note: An SDS gel was ran of the SEC fractions which corresponded to the observed peak. Western blotting was stopped at the blocking stage. Restriction Digests Ligations
Results: Figure 9
Next Steps: Western blotting will be completed tomorrow and will confirm if the bands observed on the SDS gel are indeed hHBB. The pQE80-LhHBN ligations will be transformed into M15[pREP4] E.coli tomorrrow.
Aim of experiment: To complete the Western blot of hHBB and transform pQE80-LhHBN into M15[pREP4] E.coli.
Protocols Used: Western Blotting Transformations
Results: Figure 10
Next Steps: The blot shows hHBB has been successfully purified. If the transformations are successful, overnight cultures will be set up tomorrow.
Aim of experiment: To set up overnight cultures of M15[pREP4] E.coli containing pQE80-LhHBN.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep M15[pREP4] E.coli overnight cultures containing pQE80-LhHBN.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: The miniprep with the highest concentration will be sent for sequencing.
Summary
hHBN was successfully cloned into pQE80-L and cloning started for insertion into pT25.
Aim of experiment: To digest hHBN again with BamHI and KpnI and set up ligations with pQE80-L.
Protocols Used: Resriction Digests Ligations
Results: N/A
Next Steps: Ligations will be transformed into M15[pREP4] E.coli tomorrow.
Aim of experiment: To transform M15[pREP4] E.coli with pQE80-L-hHBN.
Protocols Used: Transformations
Results: N/A
Next Steps: If transformations are successful, overnight cultures will be set up tomorrow.
Aim of experiment: To set up overnight cultures of M15[pREP4] E.coli containing pQE80-L-hHBN
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep the M15[pREP4] E.coli Overnight Cultures containing pQE80-L-hHBN.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps:The miniprep with the highest concentration will be sent for sequencing.
Summary
Cloning of hHBN into pT25 was completed this week but subsequent sequencing showed that it had not inserted successfully.
Aim of experiment: To digest the hHBN PCR product with BamHI and KpnI and set up ligations with the pT25 vector.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps:The pT25-hHBN ligations will be transformed into MG1655 E.coli tomorrow.
Aim of experiment: To set up transformations of pT25-hHBN into MG1655 E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: If transformations are successful, overnight cultures will be set up tomorrow.
Aim of experiment: To set up overnight cultures of MG1655 E.coli containing pT25-hHBN.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep the MG1655 E.coli overnight cultures containing pT25-hHBN.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: The miniprep with the highest concentration will be sent for sequencing.
Summary
Overexpression assays of hHBN were set up this week and purification of the protein was begun.
Aim of experiment: To set up overnight cultures of M15[pREP4] E.coli containing pQE80-L-hHBN.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps:The overnight cultures will be used to set up day cultures that will be induced with different concentrations of IPTG to assay hHBN expression levels.
Aim of experiment: To assay hHBN expression levels when induced with a range of IPTG concentrations.
Protocols Used: Overexpression Assay Note: Day cultures were grown to an OD600 of 0.6 and then induced with 0.25 mM, 0.5 mM, 0.75 mM and 1 mM IPTG and allowed to grow for 6 hours.
Results: SDS gel
Next Steps: The gel indicates that hHBN is not being overexpressed in any of the cultures. Further optimization experiments will be set up to try and overexpress hHBN.
Aim of experiment: To set up overnight cultures of M15[pREP4] E.coli containing pQE80-L-hHBN which will be used to set up a 3 L day culture tomorrow.
Protocols Used: Overnight Cultures Note: A 150 ml overnight culture was set up.
Results: N/A
Next Steps: The overnight cultures will be used to set up 3 L day cultures tomorrow.
Aim of experiment: To set up 3 L day cultures of M15[pREP4] E.coli containing pQE80-L-hHBN using the overnight culture set up yesterday.
Protocols Used: Protein Purification Note: The cultures were grown to OD600=0.9, then induced with 1 mM IPTG and allowed to grow for a further 8 hours. A 1 ml sample was taken before the cultures were spun down.
Results: N/A
Next Steps: Protein purification will be continued next week.
Summary
Purification of haptoglobin was completed this week and confirmed via Western Blotting.
Aim of experiment: To continue purification of hHBN by performing affinity chromatography.
Protocols Used: Protein Purification Note: The protocol was stopped at Step 13.
Results: N/A
Next Steps: The samples corresponding to the peak observed in the chromatogram will be combined and purified using SEC tomorrow.
Aim of experiment: To continue purification of hHBN by performing size exclusion chromatography.
Protocols Used: Protein Purification
Results: N/A
Next Steps: The gel shows bands corresponding to 45 kDa, the expected size of hHBN. This will be checked via Western Blotting to ensure it is the correct protein.
Aim of experiment: To perform Western Blotting in order to verify the protein that was purified is indeed hHBN.
Protocols Used: Western Blotting Note: The membrane was left blocking in milk overnight.
Results: N/A
Next Steps: The blot will be completed tomorrow.
Aim of experiment: To complete Western Blotting of hHBN.
Protocols Used: Western Blotting The protocol was continued from Step 12.
Results: Figure 14
Next Steps: The blot indicates that hHBN has been successfully purified.
Summary
Cloning of hHBA into pT25 was carried out this week.
Aim of experiment: To digest the hHBA with BamHI and KpnI and set up ligations with pT25.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps:The ligations will be left overnight and transformed into MG1065∆cya E.coli tomorrow.
Aim of experiment: To transform the pT25-hHBA ligations into MG1065∆cya E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: If transformations are successful, colonies will be pick for setting up overnight cultures tomorrow.
Aim of experiment: To set up overnight cultures of MG1065∆cya E.coli containing pT25-hHBA.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep the MG1065∆cya E.coli overnight cultures containing pT25-hHBA.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: The miniprep with highest concentration was sent for sequencing to confirm if the insert has been cloned in successfully.
Summary
Cloning of hHBN into pUT18 was carried out this week.
Aim of experiment: To digest the hHBN with BamHI and KpnI and set up ligations with pUT18.
Protocols Used: Restriction Digests Ligations
Results: N/A
Next Steps:The ligations will be left overnight and transformed into MG1065∆cya E.coli tomorrow.
Aim of experiment: To transform the pUT18-hHBN ligations into MG1065∆cya E.coli.
Protocols Used: Transformations
Results: N/A
Next Steps: If transformations are successful, colonies will be picked for setting up overnight cultures tomorrow.
Aim of experiment: To set up overnight cultures of MG1065∆cya E.coli containing pUT18-hHBN.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: The overnight cultures will be miniprepped tomorrow.
Aim of experiment: To miniprep the MG1065∆cya E.coli overnight cultures containing pUT18-hHBN.
Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit)
Results: N/A
Next Steps: The miniprep with highest concentration was sent for sequencing to confirm if the insert has been cloned in successfully.
Summary
Produced a purified plasmid of pIDT-OBP2A in order to use it as template in PCR.
Aim of Experiment: To transform pIDT-OBP2A into MC1061 e.coli strain.
Protocols Used: Transformation
Results: N/A
Next Steps: The result of this transformation can be seen tomorrow. If successful then the next step will be to produce an overnight culture in preparation of subsequent plasmid purification.
Aim of experiment: To produce an overnight culture of pIDT-OBP2A from positive colonies of MC1061 from the transformation done on the 17/6.
Protocols Used: Overnight Culture
Results: N/A
Next Steps: If the overnight culture successfully grows then the next stage of experimentation will be to produce a plasmid purification of pIDT-OBP2A from that overnight culture.
Aim of experiment: The overnight culture produced on the 18/6 will now undergo a plasmid purification in order to obtain the pIDT-OBP2A plasmid.
Protocols Used: Plasmid Purification
Results: The plasmid purification yielded a concentration of 305.53 ng/ul.
Next Steps: Assuming the plasmid purification has been successful, then the next stage of experimentation will be to run a PCR using this purified pIDT-OBP2A plasmid as the template for subsequent amplification.
Summary
Removal of the signaling peptide from OBP2A and insertion of OBP2A into the biobrick vector pSB1C3.
Aim of Experiment: To perform a PCR of the purified pIDT-OBP2A plasmid using a set of primers that have been designed to remove the signaling peptide at the start of the OBP2A gene, whilst leaving the rest of the gene intact.
Protocols Used: PCR
Results: N/A
Next Steps: If the PCR is successful, then the next step will be to gel extract OBP2A from a the gel corresponding to the size of the OBP2A gene fragment.
Aim of experiment: To perform a gel extraction of OBP2A from a PCR reaction that was performed on the 22/6. Once gel extraction has been performed a subsequent gel will be done to determine if the gel extraction had been successful.
Protocols Used: Gel Extraction
Next Steps: To perform a restriction of OBP2A in preparation for ligation.
Aim of experiment: To perform a restriction, ligation and subsequently a transformation of of the gel extracted OBP2A into the JM110 E.coli strain.
Protocols Used: Restriction Digest : Ligation : Transformation
Results: N/A
Next Steps: If the transformation is successful and positive colonies form then these colonies will then be grown overnight in preparation for plasmid purification.
Aim of experiment: The transformation performed on the 24/6 failed and as a result OBP2A will now be re-ligated into the biobrick vector pSB1C3 and subsequently transformed into the JM110 E.coli strain.
Protocols Used: Ligation: Transformation
Results: Figure 3
Next Steps:If the transformation is successful and positive colonies form then these colonies will then be grown overnight in preparation for plasmid purification.
Aim of experiment: To produce overnight cultures of pSB1C3-OBP2A from a positive colony from the transformation done on the 25/6.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Produce plasmid purifications from the overnight cultures of pSB1C3-OBP2A in preparation for presequence digest.
Aim of experiment: To perform a plasmid purification of the overnight cultures of pSB1C3-OBP2A from the 26/6. Then to subsequently perform a presequence digest of those plasmid purification.
Protocols Used: Plasmid Purification Restriction Digests
Results: Figure 4
Next Steps: It was concluded from the results of these experiments that sample 5 of pSB1C3-OBP2A will be sent for sequencing.
Summary
Positive Sequencing of pSB1C3-OBP2A and subsequent plasmid purification of pSB1C3-OBP2A.
Aim of Experiment: Sample 5 of the plasmid purified pSB1C3-OBP2A that was done on the 24/6 is sent for sequencing to determine if OBP2A has successfully ligated into pSB1C3.
Protocols Used: N/A
Results: N/A
Next Steps: If sequencing comes back positive, then the next step will be to clone OBP2A into the two vectors of the bacterial two hybrid system.
Aim of Experiment: Sequencing came back positive for pSB1C3-OBP2A. So an overnight culture of pSB1C3-OBP2A will be done in preparation for plasmid purification.
Protocols Used:Overnight Cultures
Results: N/A
Next Steps: Once the overnight culture has been given ample time to grow, the culture will then undergo a plasmid purification.
Aim of Experiment: To produce a plasmid purification of the sequenced pSB1C3-OBP2A from the overnight culture set up on the 30/6.
Protocols Used:Plasmid Purification
Results: The plasmid purified pSB1C3-OBP2A produced a concentration of 448.83 ng/ul.
Next Steps: The next stage of experimentation is to use the plasmid purification of pSB1C3-OBP2A as the template for PCR using primers that are designed to separate the gene for use in the two hybrid system.
Summary
Ran PCR of OBP2A using primers for insertion of OBP2A into pUT18 and pT25.
Aim of Experiment: To perform a PCR of OBP2A for its insertion into the bacterial two hybrid vectors; pUT18 and pT25.
Protocols Used: PCR
Results: N/A
Next Steps:The next stage of experimentation will be to gel extract and then subsequently restrict the amplified gene fragments of OBP2A.
Summary
Attempted insertion of OBP2A into the vectors for the bacterial two hybrid system; pUT18 and pT25.
Aim of Experiment: To perform a gel extraction and then subsequently a restriction of OBP2A from the PCR produced on the 17/7 using primers designed for the insertion of OBP2A into the vectors for the bacterial two hybrid system, pUT18 and pT25.
Protocols Used: Restriction Digest
Results: N/A
Next Steps: If the gel extraction and subsequent restriction is successful, then the next step will be to ligate the restricted OBP2A into the bacterial two hybrid vector, pUT18 and pT25.
Aim of Experiment: To perform a ligation and then subsequently a transformation of OBP2A into the vectors for the bacterial two hybrid system, pUT18 and pT25. Whilst using JM110 E.coli cells as a chassis for transformation.
Protocols Used: Ligation Transformation
Results: N/A
Next Steps: The results from the transformation will be seen tomorrow. If successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight colonies in preparation for purification and sequencing.
Aim of Experiment: To perform a re-ligation and transformation of OBP2A into pUT18 and pT25 - since the transformation performed on the 21/7 failed.
Protocols Used: Ligation Transformation
Results: N/A
Next Steps: If transformation is successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight colonies in preparation for purification and sequencing.
Aim of Experiment: To produce overnight cultures of pUT18-OBP2A. Unfortunately no positive colonies were produced from the transformation of OBP2A into pT25.
Protocols Used: Overnight Culture
Results: N/A
Next Steps: Once the cells have been given ample time to grow, they will undergo plasmid purification for subsequent sequencing.
Aim of Experiment: To perform a plasmid purification of the overnight cultures of pUT18-OBP2A and then a subsequent presequence digest.
Protocols Used: Plasmid Purification Restriction Digest
Results: Figure 5
Next Steps: If sequencing comes back positive for pUT18-OBP2A then this can now be implemented into the bacterial two hybrid system. Another attempt at cloning OBP2A into pT25 will be done next week.
Summary
Successful insertion of the two subunits of OBP2A into the vectors pUT18 and pT25.
Aim of Experiment: To perform a restriction digest of OBP2A for subsequent ligation into PT25. Also, sequencing came back positive for pUT18-OBP2A, so pUT18-OBP2A can now be implemented in the bacterial two hybrid system.
Protocols Used: Restriction Digests
Results: N/A
Next Steps: The next stage of experimentation will be to ligate OBP2A into the pT25 vector.
Aim of Experiment: To perform a ligation and transformation of OBP2A into the bacterial two hybrid vector pT25, so that the interaction between the two constituents of OBP2A can be observed using the bacterial two hybrid system.
Protocols Used: LigationTransformation
Results: N/A
Next Steps: If the transformation is successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight cultures of those colonies in preparation for purification and sequencing.
Aim of Experiment: To produce overnight cultures of pT25-OBP2A from the transformation done on the 30/7.
Protocols Used: Overnight Culture
Results: N/A
Next Steps: Once the cells have grown overnight they will undergo plasmid purification for subsequent sequencing.
Aim of Experiment: To perform a plasmid purification of pT25-OBP2A from the overnight cultures done on the 31/7 and then a subsequent presequence digest.
Protocols Used: Plasmid Purification Restriction Digest
Results: Figure 7
Next Steps: If sequencing comes back positive for pT25-OBP2A then this can now be implemented into the bacterial two hybrid system. The results from the sequencing should arrive early on the 3/8.
Summary
Positive sequencing of pT25-OBP2A, subsequent transformation into the bacterial two hybrid system and plating onto MacConkey agar.
Aim of Experiment: To perform a transformation of pUT18-OBP2A into BTH101, so that a subsequent transformation of pT25-OBP2A into the same chassis can occur as both plasmids are required to reside in the same chassis so that their interaction can be observed.
Protocols Used: Transformation
Results: N/A
Next Steps: If the transformation is successful, then the next stage of experimentation will be to re-transform this culture of cells using the pT25-OBP2A plasmid.
Aim of Experiment: To perform a day culture and then a subsequent transformation of the BTH101 chassis containing pUT18-OBP2A from the transformation performed on 3/8.
Protocols Used: Transformation
Results: N/A
Next Steps: If the transformation is successful then the next stage of experimentation will be to observe the protein interaction of the two subunits of OBP2A using MacConkey agar plates.
Aim of Experiment: To perform a day culture and then subsequent plating of positive colonies from the transformation performed on the 4/8. Positive colonies from this transformation contain both vectors of the bacterial two hybrid system along with the two subunits of OBP2A and so now the interaction can be observed. The day culture will be plated onto MacConkey agar which once the cells have been given enough time to grow will give a visual demonstration of the protein interaction through a color change.
Protocols Used: Transformation
Results: N/A
Next Steps: It has been decided that whilst the E.coli strain BTH101 that is being used in this experiment is useful for visualizing protein interaction, it is very unstable. A better E.coli strain called MG1655(Δcya) will be used instead as it is much more stable. This means that tomorrow overnights will be prepared of MG1655(Δcya)for transformation with pUT18-OBP2A and then subsequently pT25-OBP2A.
Aim of Experiment: To perform an overnight culture of MG1655(Δcya)E.coli in preparation for transformation with pUT18-OBP2A and then pT25-OBP2A.
Protocols Used: Overnight Culture
Results: N/A
Next Steps: Once the overnight culture has been give sufficient time to grow these cells will then be transformed with pUT18-OBP2A and then subsequently re-transformed with pT25-OBP2A.
Aim of Experiment: To perform a transformation of MG1655(Δcya)with pUT18-OBP2A. Also the results of plating of our bacterial two hybrid interaction onto MacConkey agar has produced promising results as the colonies appear to be taking up a small amount of red dye - an indicator of protein interaction. This suggests that the two separated parts of OBP2A may in fact be interacting in vivo. This interaction will need to be quantified through the use of the β-galactosidase assay.
Protocols Used: Transformation
Results: N/A
Next Steps: If the transformation of MG1655(Δcya) with pUT18-OBP2A is successful then the next step of experimentation will be re-transform positive colonies with pT25-OBP2A.
Aim of Experiment: To perform a transformation of MG1655(Δcya) containing pUT18-OBP2A with pT25-OBP2A. Positive colonies were produced from the transformation done on the 7/7 and so as a result a day culture was produced and subsequently re-transformed with pT25-OBP2A.
Protocols Used: Transformation
Results: N/A
Next Steps: If the transformation of MG1655(Δcya) with pT25-OBP2A is successful then the next step of experimentation will be to undertake the β-galactosidase assay
Summary
First attempt at determination of the protein interaction of the OBP2A subunits using the β-galactosidase assay.
Aim of Experiment: To plate control interactions that are used in the β-galactosidase assay to aid in the interpretation of the results form the assay. This first assay attempt will consist of one positive control, one negative control and one regulatory control.
Protocols Used: N/A
Results: N/A
Next Steps: The next stage of experimentation will be to perform overnight cultures from positive colonies in preparation for the β-galactosidase assay.
Aim of Experiment: To prepare overnight cultures of positive colonies of MG1655(Δcya) containing pUT18-OBP2A and pT25-OBP2A, along with the controls in preparation for the β-galactosidase assay.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Once the cells have been given sufficient time to grow then the next stage of experimentation will be to inoculate those colonies into fresh liquid broth until they reach an O.D of 0.4 before being pellet and frozen for the assay.
Aim of Experiment: To prepare cultures for use in the β-galactosidase assay, this requires a small subsample of each overnight to be inoculated into fresh LB, two done for each colony. Then left to grow until the O.D of each culture is between 0.3-0.5. Once an O.D of 0.3-0.5 has been reached then 3 replicas of 1 ml samples are taken from each culture before being spun down, the supernatant then subsequently removed and the pellet frozen.
Protocols Used: Assay Preparation
Results: N/A
Next Steps: These samples are now ready for the β-galactosidase assay.
Aim of Experiment: To determine the millers activity of the two interacting subunits of OBP2A in vivo as a basis of the level of interaction between them. This was done using the β-galactosidase assay.
Protocols Used: β-galactosidase assay
Results: Figure 8
Next Steps: As it was the first attempt at a β-galactosidase assay, subsequent assays will be performed utilizing more controls.
Aim of Experiment: To perform overnight cultures of the controls and samples that will be necessary for doing this second β-galactosidase assay. This assay will include two positive controls which are known to have a high level of interaction - these are NarG-NarJ and Zip positive controls. There will also be two negative controls, an empty MG1655(Δcya) chassis and pUT18-OBP2A with NarJ. Finally there is one regulatory control: which contains two empty vectors of pUT18 and pT25.
Protocols Used:Overnight Cultures
Results: N/A
Next Steps: The next stage of experimentation will be to prepare samples for use in the β-galactosidase assay.
Summary
Second attempt at determining the protein interactions of the two OBP2A subunits using the β-galactosidase assay and subsequent cloning of OBP2A into the bacterial two hybrid vectors in switched order.
Aim of Experiment: To prepare cultures for use in the β-galactosidase assay, this requires a small subsample of each overnight to be inoculated into fresh LB, two done for each colony. Then left to grow until the O.D of each culture is between 0.3-0.5. Once an O.D of 0.3-0.5 has been reached then 3 replicas of 1 ml samples are taken from each culture before being spun down, the supernatant then subsequently removed and the pellet frozen.
Protocols Used: β-galactosidase assay
Results: N/A
Next Steps: The next stage of experimentation will be to perform the second β-galactosidase assay on the interaction between the two subunits of OBP2A
Aim of Experiment: To perform a second attempt at the β-galactosidase assay that will aid in the determination of the level of interaction that the two separated subunits of OBP2A are inflicting on one another.
Protocols Used: β-Galactosidase assay
Results: Figure 10
Next Steps: The next stage of experimentation will be to switch the positioning of the two subunits of OBP2A in pUT18 and pT25 by having the smaller subunit in pUT18 and the large subunit in pT25. Along with this another β-galactosidase assay will be performed however this time it will be done under anaerobic conditions to see if that has any appreciable effect on the interaction of the two subunits of OBP2A.
Aim of Experiment: To Perform a PCR of OBP2A using primers for pU1T18 and pT25 insertion which have been designed to place the smaller OBP2Asubunit into pUT18 and the larger subunit into pT25.
Protocols Used: PCR
Results: N/A
Next Steps: If PCR is successful then the next stage of experimentation will be to perform a gel extraction and restriction in perpetration for ligation and transformation of OBP2A into pUT18 and pT25.
Aim of Experiment: The PCR performed on the 20/8 failed as no bands were found to be present on the gel during gel viewing. As a result of this a subsequent PCR was done today.
Protocols Used: PCR
Results: N/A
Next Steps: If PCR is successful then the next stage in experimentation will be to perform a gel extraction and restriction in perpetration for ligation and transformation of OBP2A in pUT18 and pT25.
Aim of Experiment: A second attempt at preparing OBP2A for insertion into the two vectors of the bacterial two hybrid system(pUT18 and pT25) has failed! As a result this will be left until next week, so that the rest of the week can be dedicated to preparing and undertaking another β-galactosidase assay, that will be done under anaerobic conditions.
Protocols Used: N/A
Results: N/A
Next Steps: The next stage of experimentation is to prepare for a anaerobic β-galactosidase assay of OBP2A in the bacterial two hybrid vectors; pUT18 and pT25.
Summary
Performed an anaerobic β-galactosidase assay in order to determine the level of interaction between the two subunits of OBP2A.
Aim of Experiment: To prepare the samples and controls of OBP2A for the anaerobic β-galactosidase assay, so they can be frozen overnight in preparation for the assay itself.
Protocols Used: Assay Preparation
Results:
Next Steps: The next stage of experimentation will be to perform the β-galactosidase assay itself.
Aim of Experiment: Performed an anaerobic β-galactosidase assay of OBP2A in the bacterial two hybrid vectors: pUT18 and pT25. This is to determine the level of interaction that the smaller subunit of OBP2A has with its larger subunit in vivo. This particular assay has two positive controls, two negative controls and one regulatory control.
Protocols Used: β-galactosidase assay
Results: Figure 12
Next Steps: The results have shown that the two subunits of OBP2A in fact don't seem to be interacting in vivo - a possible reason for this might be aggregation of the two subunits preventing interaction. The next stage of experimentation will be to test out the interaction when the subunit are switch in relation to heir positions on the vectors: pUT18 and pT25.
