Team:Dundee/lucylabjournal

LABJOURNAL

BioSpray

Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.

Chromium Detector

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur a tincidunt elit. Aliquam porta nibh at enim luctus, auctor consequat dolor vehicula.

Fingerprint Aging

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur a tincidunt elit. Aliquam porta nibh at enim luctus, auctor consequat dolor vehicula.

Saliva

Week Beginning 1/6/2015

Summary

Lactoferrin binding protein A (LbpA) was successfully cloned into the vector pSB1C3.

1/6: Amplification of LbpA for Cloning into pSB1C3

Aim of Experiment: Amplify LbpA from MC58 N.meningitidis template chromosome.

Protocols Used: PCR

Results: Figure 1

Next Steps: Since the LbpA gene was amplified successfully, it was ready to be digested for subsequent cloning into pSB1C3.

2/6: Restriction Digests and Ligations with 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 LbpA + pSB1C3 were left overnight until the next day when they would be transformed into the JM110 E.coli.

3/6: Transformations of pSB1C3-LpbA into 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.

4/6: Overnight Cultures of JM110 E.coli containing pSB1C3-LbpA

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 on the overnight cultures and a pre-sequence digest test performed tomorrow.

5/6: Plasmid Purification and Pre-Sequencing Digest Check

Aim of experiment: To miniprep the JM110 E.coli overnight cultures containing pSB1C3-LbpA and perform a pre-sequence restriction digest to check for the presence of the insert.

Protocols Used: Plasmid Purification (QIAprep® Spin Miniprep Kit) Restriction Digests

Results: N/A

Next Steps: Since the pre-sequencing digest worked, the miniprep will be sent for sequencing.

Week Beginning 8/6/2015

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.

8/6: Amplification of LbpA for Cloning into pQE80-L

Aim of experiment: To amplify LbpA for subsequent cloninng into the pQE80-L vector.

Protocols Used: PCR

Results: Figure 2

Next Steps: The PCR product will be digested and ligated into pQE80-L tomorrrow.

9/6: Restriction Digests and Ligations of LbpAinto pQE80-L

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.

10/6: Transformations of pQE80-L-LpbA into JM110 E.coli

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.

11/6: Colony PCR of JM110 E.coli Colonies containing pQE80-L-LbpA

Aim of experiment: To set up colony PCR from the single colony obtained from the 2:1 ligation transformation.

Protocols Used:

Results:

Next Steps: The gel indicates that the pQE80-L vector has sealed without the presence of the LbpA gene. LbpA will amplified again tomorrow.

Week Beginning 15/6/15

Summary

This week, cloning of LbpA into pQE80-L was continued due to last week's failed attempts.

15/6: PCR of LbpA for Cloning into pQE80-L

Aim of experiment: To re-amplify the LbpA gene for cloning into the pQE80-L vector.

Protocols Used: PCR

Results: Figure 3

Next Steps: The gel shows that LbpA has been successfully amplified so it will be digested and ligated into the pQE80-L vector tomorrow.

Week Beginning 22/6/15

Summary

LbpA was successfully cloned into the high expression vector pQE80-L.

23/6: Restriction Digest and Ligation of LbpA into 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 M15pREP4 E.coli.

24/6: Transformations of pQE80-L-LbpA into M15pREP4 E.coli

Aim of experiment: To transform pQE80-L-LbpA into M15pREP4 E.coli.

Protocols Used: Transformations

Results: N/A

Next Steps: Overnight cultures will be set up tomorrow if the transformations are successful.

25/6: Overnight Cultures of M15pREP4 E.coli containing pQE80-L-LbpA

Aim of experiment: To set up overnight cultures of M15pREP4 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.

26/6: Plasmid Purification of the M15pREP4 E.coli Overnight Cultures containing pQE80-L-LbpA

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.

Week Beginning 29/6/15

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.

1/7: M15pREP4 E.coli Overnight Cultures containing pQE80-L-LbpA

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.

2/7: Optimization of LbpA Expression

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.

3/7: Continuation of Optimization of LbpA Expression

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.

Week Beginning 6/7/15

Summary

Further protein expression optimization experiments were set up, this time using a range of IPTG concentrations to induce expression of LbpA.

7/7: Further Optimization of LbpA Expression

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.

Week Beginning 13/7/15

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.

14/7: Plate Reader Growth Curve Assay

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.

17/7: Sample Preparation for Western Blot

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.

Week Beginning 20/7/15

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.

20/7: Western Blot LpbA Culture Samples

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.

23/7: Growth Curve Assay

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.

Week Beginning 27/7/15

Summary

The presence of LbpA was confirmed in the culture 6 hours after induction with IPTG so protein purification was started this week.

27/7: Overnight Cultures

Aim of experiment: To set up a 150ml overnight culture for sub-culturing tomorrow.

Protocols Used: Overnight Cultures Note: 150ml of LB was used and 150µl of each antibiotic.

Results: N/A

Next Steps:150ml of fresh LB will be inoculated tomorrow with 7.5ml of the overnight cultures.

28/7: LbpA Expression Assay

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: 150ml of fresh LB was inoculated with 7.5ml of the overnight cultures. Only an uninduced control and an 1mM 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 3L culture will be set up on Thursday under the same conditions as the overnight cultures in order to try and purify LbpA.

29/7: Overnight Cultures for 3L Culture

Aim of experiment: To set up overnight cultures 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 induced with 1mM IPTG and grown for 6 hours.

30/7: 3L Culture for Purification of LbpA

Aim of experiment: To set up 3L 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.

Week Beginning 3/8/15

Summary

The first round of LbpA purification failed at the affinity chromatography stage so another 6L culture was grown this week to attempt purification again.

3/8: Continuation of LbpA Purification

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.

5/8: Overnight Cultures for 6L Culture

Aim of experiment: To set up overnight cultures for the 6L culture that will be set up tomorrow.

Protocols Used: Overnight Cultures Note: One 150ml overnight culture was set up using 150µl of each antibiotic.

Results: N/A

Next Steps:The overnight cultures will be used to set up the 6L day culture tomorrow.

6/8: 6L Day Cultures for LbpA Purification

Aim of experiment: To set up 6L 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.

Week Beginning 10/8/15

Summary

Purification of LbpA was continued but unfortunately was unsuccessful. If time permits, another round of purification may be reattempted.

10/8: Continuation of LbpA Purification

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:

11/8: Continuation of LbpA Purification

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.

Semen

Week Beginning 25/5/2015

Summary

Cloning of PotD into the biobrick vector, pSB1C3, was started this week.

28/5: PCR of PotD from MG1655 E.coli gDNA

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.

29/5: Gel Extraction, Restriction Digest and Ligation of SBP into pSB1C3

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.

Week Beginning 1/6/2015

Summary

Cloning of PotD into the biobrick vector was completed this week.

1/6: Transformation of pSB1C3 +PotD into JM110 E.coli

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.

2/6: Overnight Cultures of pSB1C3 + PotD in JM110 E.coli

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.

3/6: Plasmid Purification of pSB1C3 + PotD from Overnight Cultures

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.

Week Beginning 15/6/15

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.

15/6: PCR of PotD for Cloning into pQE80-L

Aim of experiment: To amplify the PotD gene for cloning into the pQE80-L vector.

Protocols Used: PCR

Results: Figure 3

Next Steps: The gel shows that PotD has been successfully amplified so it will be digested and ligated into the pQE80-L vector tomorrow.

16/6: Gel Extraction of PotD PCR Product

Aim of experiment: To gel extract the PotD PCR product.

Protocols Used: PCR

Results: Figure 3

Next Steps: The gel shows that PotD has been successfully amplified so it will be digested and ligated into the pQE80-L vector tomorrow.

17/6: Restriction Digest and Ligation of PotD into pQE80-L. Transformation of IDT Plasmid containing SBP into MC1061 E.coli

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:Transformations

Results: N/A

Next Steps: The PotD + pQE80-L ligations will be transformed into MC1061 E.coli. Overnight cultures of the transformations will be set up tomorrow.

18/6: Transformations of PotD + pQE80-L into MC1061 E.coli. Overnight Cultures of MC1061 E.coli with SBP + IDT Plasmid

Aim of experiment: To transform the PotD + pQE80-L 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: Figure 3

Next Steps: If transformations are successful, overnight cultures will be set up tomorrow.

19/6: Repeat of Ligation of PotD into pQE80-L. Plasmid Purification of the IDT Plasmid Containing SBP

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.

Week Beginning 22/6/15

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.

22/6: Transformation of PotD + pQE80-L Ligations. PCR and Gel Extraction of SBP for Cloning into pQE80-L and Restriction Digests and Ligation of SBP for Cloning into pSB1C3

Aim of Experiment: To transform PotD + pQE80-L 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 SBP + pSB1C3 ligations will be transformed into MC1061 E.coli.

23/6: Overnight Cultures of MC1061 E.coli containing pSB1C3 + PotD. Transformations of MC1061 E.coli with pSB1C3 + SBP. Restriction Digests and Ligations of SBP into pQE80-L and Transformations 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.

24/6: Plasmid Purification of the Overnight Cultures of MC1061 E.coli containing pSB1C3 + PotD. Overnight Cultures of the Transformations of MC1061 E.coli 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.

25/6: Plasmid Purification of the Overnight Cultures of MC1061 E.coli containing pSB1C3 + SBP and pQE80-L + SBP

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.

Week Beginning 29/6/15

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.

29/6: Ligations of SBP into pSB1C3 and Transformations into MC1061 E.coli

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.

30/6: Overnight Cultures of MC1061 E.coli containing pSB1C3 + SBP, pQE80-L + SBP and pQE80-L + PotD

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.

1/7: Plasmid Purification of the Overnight Cultures of MC1061 E.coli containing pSB1C3 + SBP. PotD and SBP Expression Optimization

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.

2/7: Continuation of PotD and SBP Expression Optimization

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.

3/7: Western Blot of PotD and SBP Day Culture Samples

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.

Week Beginning 6/7/15

Summary

Further protein expression optimization experiments were set up, this time using a range of IPTG concentrations to induce expression of SBP.

6/7: Further Optimization of SBP Expression

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 o.5mM, 1mM and 2mM IPTG. The Western Blot was left to block overnight.

Results: Figure 4

Next Steps: The Western Blot will be completed tomorrow.

7/7: Western Blot of SBP

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.

Week Beginning 13/7/15

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.

14/7: Plate Reader Growth Curve Assay

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.

15/7: Overnight Cultures for 3L Culture for PotD Purification

Aim of experiment: To set up overnight cultures that will be used tomorrow to set up 3L cultures for PotD purification.

Protocols Used: Overnight Cultures

Results: N/A

Next Steps: The 3L day culture will be set up tomorrow.

16/7: Sample Preparation for Western Blot of SBP and 3L Day Culture for PotD Purification

Aim of experiment: To check if the cells in the induced cultures ~7 hours after being induced are producing SBP. To set up 3L 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.

17/7: Harvesting PotD Cultures

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.

Week Beginning 20/7/15

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.

20/7: Western Blot SBP Culture Samples

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 150ml overnight culture was set up for PotD so 3L cultures could be set up tomorrow.

21/7: 3L Day Cultures for PotD Purification

Aim of experiment: To set up 3L 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.

22/7: Continuation of PotD Purification

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.

23/7: Continuation of PotD Purification and Growth Curve Assay for SBP

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.

24/7: SDS-PAGE and Western Blot of PotD

Aim of experiment: To run SDS-PAGE of the elutions obtained on Friday after SEC.

Protocols Used: SDS-PAGE

Results: Figure 7

Next Steps:The same samples will be Western Blotted on Monday to confirm that the bands oberved are PotD.

Week Beginning 27/7/15

Summary

PotD was successfully purified and characterized this week. SBP was still unobservable in induced 150ml cultures so 3L cultures containing pQE80-L-SBP were set up.

27/7: Overnight Cultures for SBP Expression Assays and SDS-PAGE and Western Blot of PotD

Aim of experiment: To set up a 150ml 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: 150ml of LB was used and 150µl of each antibiotic. PotD: SDS-PAGE and Western Blotting Note: The membrane was left blocking overnight.

Results: N/A

Next Steps:150ml of fresh LB will be inoculated tomorrow with 7.5ml of the overnight cultures containing pQE80-L-SBP. The PotD Western Blot will be completed tomorrow.

28/7: SBP Expression Assay and Completion of PotD Westen Blot

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: 150ml of fresh LB was innoculated with 7.5ml of the overnight cultures. Only an uninduced control and an 1mM 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, 3L cultures will be set up this week to try and harvest the protein since the culture size may be too small.

29/7: Overnight Cultures for 3L Culture

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.

30/7: 3L Culture for Purification of SBP

Aim of experiment: To set up 3L day cultures for purifying SBP.

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.

Week Beginning 3/8/15

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. .

3/8: Continuation of SBP Purification

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.

4/8: Continuation of SBP Purification

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.

6/8: Western Blot of SBP

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.

Week Beginning 10/8/15

Summary

Interactions between PotD and spermidine were analysed via tryptophan fluorescence this week.

10/8: Tryptophan Fluorescence

Aim of experiment: To analyse the interaction of PotD and spermidine using tryptophan fluorescence.

Protocols Used: Tryptophan Fluorescence

Results: N/A

Next Steps:

Blood

Week Beginning 10/6/2015

Summary

Cloning of hHBA and hHBB

into the biobrick vector, pSB1C3, was performed this week.

10/6: Restriction Digests of hHBA and hHBB from IDT gBlocks and Ligation into pSB1C3

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.

11/6: Transformations of pSB1C3-hHBA and pSB1C3-hHBB Ligations 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.

Week Beginning 15/6/2015

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.

15/6: Overnight Cultures of JM110 E.coli containing pSB1C3-hHBA and pSB1C3-hHBB

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.

16/6: Plasmid Purification of the JM110 E.coli Overnight Cultures containing pSB1C3-hHBA and pSB1C3-hHBB

Aim of experiment: To miniprep the JM110 E.coli Overnight Cultures containing pSB1C3-hHBA and pSB1C3-hHBB

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.

17/6: Amplification of hHBA and hHBB for Cloning into the Two Hybrid System Vectors

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 1bp 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.

18/6: Gel Extraction and Restriction Digests hHBB for Cloning into pUT18

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.

19/6: Ligations of hHBB into pUT18

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.

Week Beginning 22/6/15

Summary

Cloning of hHBA and hHBB into pT25 and pUT18, respectively, was continued this week.

22/6: Transformations of pUT18-hHBB into BTH101 E.coli

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.

23/6: Overnight Cultures of BTH101 E.coli containing pUT18-hHBB

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.

24/6: Plasmid Purification of the BTH101 E.coli Overnight Cultures containing pUT18-hHBB

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.

25/6: Amplification of hHBA for Cloning into pT25

Aim of experiment: To amplify hHBA for Cloning into pT25

Protocols Used: PCR

Results: Figure 3

Next Steps: The PCR product digested and ligated into pT25 tomorrow.

26/6: Restriction Digests and Ligations of hHBA 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.

Week Beginning 29/6/15

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.

29/6: Transformation of pT25-hHBA Ligations into BTH101 E.coli/span>

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.

30/6: Overnight Cultures of MC1061 E.coli containing pUT18-hHBA and Amplification of hHBA and hHBB for Cloning into pQE80-L

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 PCR

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.

1/7: Plasmid Purification of the Overnight Cultures of BTH101 E.coli containing pT25-hHBA and Restriction Digests and Ligations of hHBA and hHBB into pQE80-L

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.

2/7: Transformations of pQE80-L-hHBA and pQE80-L-hHBB Ligations into M15[pREP4] E.coli

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.

Week Beginning 6/7/15

Summary

Cloning of hHBA and hHBB into pQE80-L was completed this week. Cloning of Haptoglobin (hHBN) was also started this week.

6/7: Overnight Cultures of m15[pREP4] E.coli containing pQE80-L-hHBA and pQE80-L-hHBB

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.

7/7: Plasmid Purification of m15[pREP4] E.coli Overnight Cultures containing pQE80-L-hHBA and pQE80-L-hHBB

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.

8/7: Transformation of IDT Plasmid-hHBN into MC1061 E.coli

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.

9/7: Overnight Cultures of MC1061 E.coli containing IDT Plasmid-hHBN

Aim of experiment: To set up overnight cultures of MC1061 E.coli containing IDT Plasmid-hHBN.

Results: N/A

Next Steps: The overnight cultures will be miniprepped tomorrow and hHBN will be amplified for cloning into pSB1C3.

10/7: Plasmid Purification of IDT Plasmid-hHBN and Digestion of hHBN 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.

Week Beginning 13/7/15

Summary

hHBB was successfully characterized this week so protein purification was started. Cloning of hHBN into pSB1C3 was also completed.

13/7: Ligations of hHBN into pSB1C3 and Overnight Cultures of M15[pREP4] E.coli containing pQE80-L-hHBA and pQE80-L-hHBB

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.

14/7: Transformation of pSB1C3-hHBN into MC1061 E.coli and Optmiization of hHBA and hHBB Expression

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.

15/7: Overnight Cultures of MC1061 E.coli containing pSB1C3-hHBN and Western Blotting of 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. 3L cultures for purification of hHBB will be set up this week. Characterization of hHBA may be reattempted. The overnight cultures will be miniprepped tomorrow.

16/7: Plasmid Purification of pSB1C3-hHBN and Overnight Cultures of M15[pREP4] E.coli containing pQE80-L-hHBB

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: Figure 7

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.

17/7: 3L Cultures of M15[pREP4] E.coli containing pQE80-L-hHBB

Aim of experiment: To set up 3L 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.

Week Beginning 20/7/15

Summary

hHBB was successfully purified this week and cloning of hHBN into pQE80-L was completed.

20/7: Amplification of hHBN for Cloning into pQE80-L and Purification of hHBB

Aim of experiment: To amplify hHBN for cloning into pQE80-L and continuation of hHBB purification.

Protocols Used: PCR Protocols Used: Protein Purification Note: Protocol was continued from Step 6 PCR

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.

21/7: Size Exclusion Chromatography of hHBB and Restriction Digests and Ligations of hHBN into pQE80-L

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.

22/7: Western Blot of hHBB and Transformations of m15[pREP4] E.coli with pQE80-LhHBN Ligations

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.

23/7: Overnight Cultures of m15[pREP4] E.coli containing pQE80-LhHBN

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.

24/7: Plasmid Purification of Overnight Cultures of m15[pREP4] E.coli containing pQE80-LhHBN

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.

Week Beginning 27/7/15

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.

27/7: Restriction Digests and Ligations of hHBN into pQE80-L.

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.

28/7: Transformation of M15[pREP4] E.coli with pQE80-L-hHBN

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.

29/7: Overnight Cultures of M15[pREP4] E.coli containing pQE80-L-hHBN

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.

30/7: Plasmid Purification of M15[pREP4] E.coli Overnight Cultures containing pQE80-L-hHBN

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.

31/7: Amplification of hHBN for Cloning into pT25

Aim of experiment: To amplfy hHBN for cloning into the two hybrid vector pT25.

Protocols Used: PCR

Results: Figure 11

Next Steps:The PCR product will be digested and ligated into pT25 on Monday.

Week Beginning 3/8/15

Summary

PotD was successfully purified and characterized this week. SBP was still unobservable in induced 150ml cultures so 3L cultures containing pQE80-L-SBP were set up.

27/7: Overnight Cultures for SBP Expression Assays and SDS-PAGE and Western Blot of PotD

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.

4/8: Transformations of pT25-hHBN into MG1655 E.coli

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.

5/8: Overnight Cultures of MG1655 E.coli containing pT25-hHBN

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.

6/8: Plasmid Purification of MG1655 E.coli Overnight Cultures containing pT25-hHBN

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.

Week Beginning 10/7/15

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. .

10/8: Overnight Cultures for hHBN Overexpression Assays

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.

11/8: Overexpression Assay of pQE80-L-hHBN

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.

12/8: Overnight Cultures of M15[pREP4] E.coli containing pQE80-L-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 3L day culture tomorrow.

Protocols Used: Overnight Cultures Note: A 150ml overnight culture was set up.

Results: N/A

Next Steps: The overnight cultures will be used to set up 3L day cultures tomorrow.

13/8: 3L Day Cultures of M15[pREP4] E.coli containing pQE80-L-hHBN

Aim of experiment: To set up 3L 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.

Week Beginning 17/8/15

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. .

17/8: hHBN Purification

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.

18/8: hHBN Purification

Aim of experiment: To continue purification of hHBN by performing size exclusion chromatography.

Protocols Used: Protein Purification

Results:

The gel shows bands corresponding to 45kDa, the expected size of hHBN. This will be checked via Western Blotting to ensure it is the correct protein.

19/8: Western Blotting of Purified hHBN

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.

20/8: Western Blotting of Purified hHBN

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: Protein purification will be continued next week.

×

PCR Protocol

PCR reaction mixtures were set up according to the table below. The length of all sequences used is stated in the Sequences section.

Reactants	Volume (µl)
DNA	1
DMSO	2.5
Forward Primer	0.5
Reverse Primer	0.5
Herculase Buffer	10
dNTP	0.5
Herculase	0.5
H2O	34.5

**PCR Programme**

Elongation time was altered according to the length of the sequences.

Close

×

Restriction Digests Protocol

Restriction digests were set up according to the table below. The appropriate reaction and restriction enzymes that were used is stated in the Lab Book section.

	Post Plasmid Purification	Post PCR	gBlock
DNA	1mg	50µl	100ng
Cutsmart Buffer (µl)	6	6	6
Water (µl)	As required	1	As required
Enzyme 1 (µl)	1.5	1.5	1.5
Enzyme 2 (µl)	1.5	1.5	1.5
Total Volume (µl)	30	60	30

The digests were incubated at 37^oC for 3 hours.
Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of Cutsmart Buffer was added at the 2 hour and 2.5 hour mark.

Close

×

Ligation Protocol

Ligation reactions were set up according to the table below. The vector to insert ratio is stated for each specific reaction in the Lab Book section.

	Vector (µl)	2:1 (Vector: Insert) [µl]	3:1 (Vector: Insert) [µl]	4:1 (Vector: Insert) [µl]
T4 DNA Ligase	1	1	1	1
Ligase Buffer	1	1	1	1
Vector	1	1	1	1
Insert	-	2	3	4
H20	7	5	4	3

The reactions were incubated at room temperature for at least 3 hours.

Close

×

Transformation Protocol

1. 100µl of competent cells were defrosted. Thaw on ice.

2. Agar plates were taken out of 4^oC to warm to room temperature.

3. 1-5µl of DNA was added to cells.

4. Cell and DNA mixture was left on ice for 20 minutes.

5. Mixture was placed in 42^oC water bath for 90 seconds.

6. Mixture was put back on ice for 2 minutes.

7. 1ml of LB (without antibiotic) was added to cells.

8. Cells were allowed to grow for 1 hour at 37^oC in a shaking incubator.

9. Cells were then spun down and the supernatant discarded.

10. The pellet was resuspended in the 100µl.

11. Cells were plated on agar plates with appropriate antibiotic.

Close

×

Miniprep Protocol

1. 1-5ml bacterial overnight culture was pelleted by centrifugation at >8000rpm for 3 minutes at room temperature.

2. Pelleted bacterial cells were resuspended in 250µl of Buffer P1 and transferred to a microcentrifuge tube.

3. 250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution becomes clear.

4. 350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.

5. The mixture was centrifuged for 10 minutes at 13000rpm in a microcentrifuge.

6. The supernatant was applied to a QIAprep spin column by pipetting.

7. The QIAprep spin column was washed with 500µl of Buffer PB by centrifugation for 1 minute and flow through discarded.

8. 750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute and flow through discarded.

9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer.

10. The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 50µl of H₂O to the QIAprep spin column and centrifuging for 1 minute.

Close

×

Overnight Culture Protocol

1. Four colonies were picked from the desired plate.

2. For each colony, 5ml of fresh LB plus the appropriate antibiotics was inoculated and grown overnight at 37^oC.

Close

×

SDS-PAGE and Western Blotting

1. 50µl of an overnight culture was used to inoculate 5ml of fresh LB containing the appropriate antibiotics and left to grow at 37^oC in a shaking incubator.

2. Once the OD₆₀₀=0.6-0.8, 1mM of IPTG was added to the culture and left to grow for a further 3 hours.

3. 1ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.

4. Meanwhile, 950µl of 2x Laemmli sample buffer was added to 50µl of B-mercaptoethanol.

5. The supernatant was discarded from the samples and the pellet resuspended in 200µl of the above solution.

6. The samples were then boiled at 90^oC for 10 minutes.

7. Afterwards, they were spun down for 1 minute in a microcentrifuge.

8. At this stage, the samples could be stored at -20^oC or ran on a gel immediately.

9. Two gradient gels consisting of a 12% separating gel and a stacking gel were prepared and the samples ran at 100V until proteins reach end of the stacking gel at which point the voltage was increased to 200V for 20 minutes.

10. One gel was stained using Instant Blue and the other was used to transfer the proteins onto a membrane.

11. After the proteins had been transferred onto the membrane using a semi dry transfer ran for 40 minutes at 5V, the membrane was blocked in 5% Marvel milk overnight at 4^oC.

12. The next day, the membrane was washed 3 times in 1xTBST buffer and then suspended in 10ml of 1xTBS buffer to which the primary anti-His antibody was added and left for one hour. The protein ladder was cut off before adding the primary antibody.

13. The membrane was then washed 3 times in 1xTBST buffer for 5 minutes and then suspended in 10ml of 1xTBS buffer, to which the secondary antibody was added and left for one hour.

14. The membrane was then washed in 3x10ml 1xTBST buffer for 5 minutes.

15. The blot was then soaked in developing solution for 5 minutes before being wrapped in a plastic cover aligned with the protein ladder that was cut off at step 12.

16. Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer.

Close

×

Growth Curve Assay Protocol

1. 5ml overnight cultures of the appropriate strains were set up.

2. The next day, 4x 995µl of plain LB containing 1µl ampicillin and 1µl of chloramphenicol were inoculated with 5µl of the overnight culture.

3. Three were induced with 0.5mM, 1mM and 2mM of IPTG and one was left uninduced.

4. 200µl of each culture was loaded onto a 96 well microtitre plate with 4 repeats.

5. The cultures were left to grow for 16 hours in the plate reader which took OD readings every 10 minutes.

Close

×

Growth Curve Assay Protocol

1. 5ml overnight cultures of the appropriate strains were set up.

2. The next day, 4x 995µl of plain LB containing 1µl ampicillin and 1µl of chloramphenicol were inoculated with 5µl of the overnight culture.

3. 20 x 200µl aliquots were loaded onto a 96 well microtitre plate.

4. After the OD₆₀₀=~0.6, three cultures were induced with 0.5mM, 1mM and 2mM of IPTG and one was left uninduced (with 4 repeats).

5. The cultures were left to grow for 20 hours in the plate reader which took OD readings every 30 minutes.

Close

×

Protein Purification Protocol

1. A 150ml overnight culture was set up for the desired protein.

2. 3 x 1L of fresh LB containing 1ml of appropriate antibiotics was inoculated with each 50ml overnight culture and left to grow until an OD₆₀₀ =0.6-1 in a shaking incubator at 37^oC.

3. The cultures were subsequently induced with 1mM IPTG and left to grow for 6 hours in a shaking incubator at 20^oC.

4. They were then centrifuged for 20 minutes at 4200rpm at 4^oC.

5. The pellets were then resuspended in 5ml of ice cold 50mM 7.5 Tris and transferred to a 50ml falcon tube and then topped up to 40ml with 50mM 7.5 Tris.

6. They were then spun down again at 4000rpm for 15 minutes at 4^oC, the supernatant discarded and pellets stored at -20^oC.

7. Once purification could proceed, the pellets were thawed on ice and resuspended in 40ml of 50mM Tris containing DNAase, 1 x protease inhibitor tablet and lysozyme. (Note: If purifying a membrane protein, 1% DDM was also added).

8. The cells were lysed using a cell disruptor.

9. Afterwards the lysate was spun down in a J25.50 rotor at 15000rpm at 4^oC for 40 minutes.

10. The supernatant was removed and transferred to a 50ml falcon tube. The pellet was kept for testing. Both were kept on ice.

11. Using FPLC, the supernatant was loaded onto a Ni²⁺ column.

12. The protein was eluted using an increasing concentration of imidazole. The two buffers used were Buffer A: 50mM Tris, 200mM NaCl, 20mM imidazole and Buffer B: 50mM Tris, 200mM NaCl, 1M Imidazole. (Note: If purifying a membrane protein 0.02% DDM was added to these buffers.)

13. The fractions corresponding to any peaks were retained and concentrated using an appropriate filter size. A small amount of each fraction could be ran on an SDS gel at this stage.

14. Using FPLC, the sample was then loaded onto a SEC column and any fractions corresponding to a peak were ran on an SDS gel to check for the presence of the desired protein.

15. Fractions containing the desired protein were collated, to which 20% glycerol was added.

16. The samples were then flash frozen and stored at -80^oC.

Close

×

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

'

Close

×

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

'

Close

×

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

'

Close

×

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.

Close

×

Figure 5: OD₆₀₀ Readings from Uninduced Culture and Induced Cultures (0.5mM, 1mM and 2mM 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.

Close

×

Figure 6: Growth Curve of Uninduced and Induced Cultures (0.5mM, 1mM and 2mM 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.

Close

×

Figure 7: Growth Curve of Uninduced and Induced Cultures (0.5mM, 1mM and 2mM 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.

Close

×

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

Close

×

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

Close

×

Figure 2: SDS Gel of Uninduced and 1mM 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 37kDa 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.

Close

×

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 1mM 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.

Close

×

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.

Close

×

Figure 5: Growth Curve of Uninduced and Induced Cultures (0.5mM, 1mM and 2mM 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.

Close

×

Figure 6: Growth Curve of Uninduced and Induced Cultures (0.5mM, 1mM and 2mM 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.

Close

×

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

Close

×

Figure 8: Characterisation of PotD following Purification. 10µl of each fraction was mixed with 10µl of 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.

Close

×

Figure 9: Characterisation of SBP following Purification. 10µl of each fraction was mixed with 10µl of 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.

Close

×

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.

Close

×

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.

Close

×

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.

Close

×

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.

Close

×

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.

Close

×

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 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.

Close

×

Figure 7: Characterisation of hHBB. Single colonies of M15[pREP4] E.coli containing pQE80-L encoding hHBB 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 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. 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.

Close

×

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 1200bp.

Close

×

Figure 9: SDS-PAGE of Fractions Obtained After Size Exclusion Chromatography of hHBB 10µl of each fraction was mixed with 10µl 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 16kDa. Western Blotting will confirm if these proteins are hHBB.

Close

×

Figure 10: Characterisation of hHBB following Purification. 10µl of each fraction was mixed with 10µl of 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.

Close

×

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 1200bp.

Close