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Revision as of 18:30, 10 August 2015
LABJOURNAL
BioSpray
Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.
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 LbpA + pSB1C3 were left overnight until the next day when they would be transformed into the JM110 strain of E.coli.
Aim of experiment: To transform the pSB1C3 + LbpA ligations into JM110 E.coli cells.
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 the LbpA + pSB1C3 JM110 colonies.
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.
Aim of experiment: To miniprep the LbpA + pSB1C3 JM110 overnight cultures 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.
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 restrict and ligate the LbpA gene into the pQE80-L plasmid.
Protocols Used: Restriction Digests Ligations
Results:: N/A
Next Steps: Transformations of LbpA + pQE80-L into the JM110 strain will be carried out tomorrow.
Aim of experiment: To transform the LbpA + pQE80-L ligations into the E.coli strain JM110.
Protocols Used: Transformations
Results: N/A
Next Steps: If the transformations are successful, colonies will be picked to set up overnight cultures.
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.
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 the M15pREP4 strain of E.coli.
Aim of experiment: To transform the ligations of LbpA+ pQE80-L into the m15pREP4 strain of 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 the colonies obtained from the LbpA + pQE80-L transformations.
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 -20oC 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 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.
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: 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.
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.
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 -20oC.
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 6L 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 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.
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.
Summary
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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:
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