Revision as of 17:01, 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.

Chromium Detector

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Fingerprint Aging

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Week Beginning 1/6/2015

Summary

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

1/6: Amplification of LbpA

Aim of Experiment: Amplify LbpA from 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

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.

3/6: Transformations of LpbA + pSB1C3 into JM110 E.coli Strain

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.

4/6: Overnight Cultures

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.

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

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.

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 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 LbpA + pQE80-L into the JM110 strain will be carried out tomorrow.

10/6: Transformations of LbpA + pQE80-L into JM110

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.

11/6: Colony PCR of LbpA + pQE80-L Transformation

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 the M15pREP4 strain of E.coli.

24/6: Transformations of LbpA + pQE80-L into 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.

25/6: Overnight Cultures of LbpA + pQE80-L Transformations

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.

26/6: Plasmid Purification of the LbpA + pQE80-L Overnight Cultures

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: LbpA + pQE80-L Overnight Cultures

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 -20oC 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 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.

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

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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:

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

	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

	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

@@ Line 70: / Line 70: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">1/6: Amplification of LbpA</span>
+             <span class="box-title">1/6: Amplification of <i>LbpA</i></span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-1"></span>
                  <div id="collapsiblew1-1" class="collapse box-content">
@@ Line 76: / Line 76: @@
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR</a></p>
           <p><b>Results: </b><a data-toggle="modal" data-target="#lbpa-figure1-modal" class="journal-protocol"> Figure 1 </a></p>
-                   <p><b>Next Steps:</b> Since the LbpA gene was amplified successfully, it was ready to be digested for subsequent cloning into pSB1C3.</p>
+                   <p><b>Next Steps:</b> Since the <i>LbpA</i> gene was amplified successfully, it was ready to be digested for subsequent cloning into pSB1C3.</p>
                  </div>
          </div>
@@ Line 89: / Line 89: @@
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-2"></span>
                  <div id="collapsiblew1-2" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To digest LbpA with BamHI and EcoRI and then ligate it into pSB1C3.</p>
+                   <p><b>Aim of experiment:</b> To digest <i>LbpA</i> with BamHI and EcoRI and then ligate it into pSB1C3.</p>
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol">Restriction Digests</a> <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations</a></p>
           <p><b>Results:</b> N/A
-                   <p><b>Next Steps:</b> Ligations of LbpA + pSB1C3 were left overnight until the next day when they would be transformed into JM110 cells.
+                   <p><b>Next Steps:</b> Ligations of <i>LbpA</i> + pSB1C3 were left overnight until the next day when they would be transformed into the JM110 strain of <i>E.coli</i>.
                  </div>
          </div>
@@ Line 104: / Line 104: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">3/6: Transformations of LpbA + pSB1C3 into JM110 </span>
+             <span class="box-title">3/6: Transformations of <i>LpbA</i> + pSB1C3 into JM110 <i>E.coli</i> Strain </span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-3"></span>
                  <div id="collapsiblew1-3" class="collapse box-content">
-                  <p><b>Aim of experiment:</b> To transform the pSB1C3 + LbpA ligations into JM110 cells.</p>
+                  <p><b>Aim of experiment:</b> To transform the pSB1C3 + <i>LbpA</i> ligations into JM110 <i>E.coli</i> cells.</p>
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformations </a></p>
                    <p><b>Results:</b> N/A
@@ Line 125: / Line 125: @@
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-4"></span>
                  <div id="collapsiblew1-4" class="collapse box-content">
-                  <p><b>Aim of experiment:</b> To set up overnight cultures of the LbpA + pSB1C3 JM110 colonies.</p>
+                  <p><b>Aim of experiment:</b> To set up overnight cultures of the <i>LbpA</i> + pSB1C3 JM110 colonies.</p>
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol"> Overnight Cultures
            </a></p>
@@ Line 143: / Line 143: @@
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-5"></span>
                  <div id="collapsiblew1-5" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To miniprep the LbpA + pSB1C3 JM110 overnight cultures and perform a pre-sequence restriction digest to check for the presence of the insert.</p>
+                   <p><b>Aim of experiment:</b> To miniprep the <i>LbpA</i> + pSB1C3 JM110 overnight cultures and perform a pre-sequence restriction digest to check for the presence of the insert.</p>
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid Purification (QIAprep® Spin Miniprep Kit) </a> <a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol"> Restriction Digests
            </a></p>
@@ Line 170: / Line 170: @@
-          <p class="journal-content">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.</p>
+          <p class="journal-content">Sequencing confirmed that <i>LbpA</i> had been successfully cloned into pSB1C3 therefore this week cloning was attempted to put <i>LbpA</i> into the high expression vector pQE80-L.</p>
           </div>
           <div id="collapsiblew2" class="collapse week-content">
@@ Line 178: / Line 178: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">8/6: Amplification of LbpA into pQE80-L</span>
+             <span class="box-title">8/6: Amplification of <i>LbpA</i> into pQE80-L</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2-1"></span>
                  <div id="collapsiblew2-1" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To amplify LbpA for subsequent cloninng into the pQE80-L vector.</p>
+                   <p><b>Aim of experiment:</b> To amplify <i>LbpA</i> for subsequent cloninng into the pQE80-L vector.</p>
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR </a></p>
                    <p><b>Results:</b><a data-toggle="modal" data-target="#lbpa-figure2-modal" class="journal-protocol"> Figure 2 </a></p>
@@ Line 194: / Line 194: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">9/6: Restriction Digests and Ligations of LbpA into pQE80-L</span>
+             <span class="box-title">9/6: Restriction Digests and Ligations of <i>LbpA</i>into pQE80-L</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2-2"></span>
                  <div id="collapsiblew2-2" class="collapse box-content">
-                   <p><b>Aim of experiment:</b>To restrict and ligate the LbpA gene into the pQE80-L plasmid.</p>
+                   <p><b>Aim of experiment:</b>To restrict and ligate the <i>LbpA</i> gene into the pQE80-L plasmid.</p>
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol">Restriction Digests </a> <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations </a> </p>
                <p><b> Results:</b>: N/A </p>
-<p><b> Next Steps:</b> Transformations of LbpA + pQE80-L into the JM110 strain will be carried out tomorrow.</p>
+<p><b> Next Steps:</b> Transformations of <i>LbpA</i> + pQE80-L into the JM110 strain will be carried out tomorrow.</p>
                  </div>
          </div>
@@ Line 210: / Line 210: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">10/6: Transformations of LbpA + pQE80-L into JM110</span>
+             <span class="box-title">10/6: Transformations of <i>LbpA</i> + pQE80-L into JM110</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2-3"></span>
                  <div id="collapsiblew2-3" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To transform the LbpA + pQE80-L ligations into the E.coli strain JM110.</p>
+                   <p><b>Aim of experiment:</b> To transform the <i>LbpA</i> + pQE80-L ligations into the E.coli strain JM110.</p>
                    <p><b>Protocols Used:</b> <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformations </a></p>
                <p><b>Results:</b> N/A </p>
@@ Line 226: / Line 226: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">11/6: Colony PCR of LbpA + pQE80-L Transformation</span>
+             <span class="box-title">11/6: Colony PCR of <i>LbpA</i> + pQE80-L Transformation</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2-4"></span>
                  <div id="collapsiblew2-4" class="collapse box-content">
@@ Line 232: / Line 232: @@
                    <p><b>Protocols Used: </b></p>
                    <p><b>Results: </b> </p>
-<p><b>Next Steps:</b> The gel indicates that the pQE80-L vector has sealed without the presence of the LbpA gene. LbpA will amplified again tomorrow. </p>
+<p><b>Next Steps:</b> The gel indicates that the pQE80-L vector has sealed without the presence of the <i>LbpA</i> gene. <i>LbpA</i> will amplified again tomorrow. </p>
 </div>
          </div>
@@ Line 257: / Line 257: @@
-          <p class="journal-content">This week, cloning of LbpA into pQE80-L was continued due to last week's failed attempts.</p>
+          <p class="journal-content">This week, cloning of <i>LbpA</i> into pQE80-L was continued due to last week's failed attempts.</p>
           </div>
@@ Line 266: / Line 266: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">15/6: PCR of LbpA for Cloning into pQE80-L</span>
+             <span class="box-title">15/6: PCR of <i>LbpA</i> for Cloning into pQE80-L</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-1"></span>
                  <div id="collapsiblew3-1" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To re-amplify the LbpA gene for cloning into the pQE80-L vector.</p>
+                   <p><b>Aim of experiment:</b> To re-amplify the <i>LbpA</i> gene for cloning into the pQE80-L vector.</p>
                    <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR</a></p>
                    <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure3-modal" class="journal-protocol">Figure 3</a></b> </p>
-<p><b>Next Steps:</b> The gel shows that LbpA has been successfully amplified so it will be digested and ligated into the pQE80-L vector tomorrow.  </p>
+<p><b>Next Steps:</b> The gel shows that <i>LbpA</i> has been successfully amplified so it will be digested and ligated into the pQE80-L vector tomorrow.  </p>
 </div>
          </div>
@@ Line 297: / Line 297: @@
-          <p class="journal-content">LbpA was successfully cloned into the high expression vector pQE80-L.</p>
+          <p class="journal-content"><i>LbpA</i> was successfully cloned into the high expression vector pQE80-L.</p>
           </div>
@@ Line 306: / Line 306: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">23/6: Restriction Digest and Ligation of LbpA into pQE80-L</span>
+             <span class="box-title">23/6: Restriction Digest and Ligation of <i>LbpA</i> into pQE80-L</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-1"></span>
                  <div id="collapsiblew4-1" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To digest the amplified LbpA gene with BamHI and KpnI and subsequently ligate it into the pQE80-L vector.</p>
+                   <p><b>Aim of experiment:</b> To digest the amplified <i>LbpA</i> gene with BamHI and KpnI and subsequently ligate it into the pQE80-L vector.</p>
                    <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#restrictiondigest-modal" class="journal-protocol"> Restriction Digests </a> <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligations</a></p>
                    <p><b>Results:</b> N/A </p>
-<p><b>Next Steps:</b> The ligations will be transformed into the M15pREP4 strain of E.coli. </p>
+<p><b>Next Steps:</b> The ligations will be transformed into the M15pREP4 strain of <i>E.coli</i>. </p>
 </div>
@@ Line 322: / Line 322: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">24/6: Transformations of LbpA + pQE80-L into E.coli</span>
+             <span class="box-title">24/6: Transformations of <i>LbpA</i> + pQE80-L into E.coli</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-2"></span>
                  <div id="collapsiblew4-2" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To transform the ligations of LbpA + pQE80-L into the m15pREP4 strain of E.coli.</p>
+                   <p><b>Aim of experiment:</b> To transform the ligations of <i>LbpA</i>+ pQE80-L into the m15pREP4 strain of <i>E.coli.</i></p>
                    <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformations</a></p>
                    <p><b>Results:</b> N/A </p>
@@ Line 338: / Line 338: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">25/6: Overnight Cultures of LbpA + pQE80-L Transformations</span>
+             <span class="box-title">25/6: Overnight Cultures of <i>LbpA</i> + pQE80-L Transformations</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-3"></span>
                  <div id="collapsiblew4-3" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To set up overnight cultures of the colonies obtained from the LbpA + pQE80-L transformations.</p>
+                   <p><b>Aim of experiment:</b> To set up overnight cultures of the colonies obtained from the <i>LbpA</i> + pQE80-L transformations.</p>
                    <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol">Overnight Cultures </a></p>
                    <p><b>Results:</b> N/A </p>
-<p><b>Next Steps:</b> Overnight cultures will be miniprepped and a pre-sequence restriction digest will be performed to check for the presence of the LbpA gene. </p>
+<p><b>Next Steps:</b> Overnight cultures will be miniprepped and a pre-sequence restriction digest will be performed to check for the presence of the <i>LbpA</i> gene. </p>
 </div>
@@ Line 353: / Line 353: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">26/6: Plasmid Purification of the LbpA + pQE80-L Overnight Cultures</span>
+             <span class="box-title">26/6: Plasmid Purification of the <i>LbpA</i> + pQE80-L Overnight Cultures</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-4"></span>
                  <div id="collapsiblew4-4" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> 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.</p>
+                   <p><b>Aim of experiment:</b> To miniprep the overnight cultures from yesterday and perform a pre-sequencing restriction digest using BamHI and KpnI to check for presence of <i>LbpA</i> gene.</p>
                    <p><b>Protocols Used: </b><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid Purification (QIAprep® Spin Miniprep Kit)</a></p>
                    <p><b>Results:</b> N/A </p>
-<p><b>Next Steps:</b> A sample of the miniprep was sent for sequencing to confirm the presence of LbpA in the pQE80-L vector. </p>
+<p><b>Next Steps:</b> A sample of the miniprep was sent for sequencing to confirm the presence of <i>LbpA</i> in the pQE80-L vector. </p>
 </div>
@@ Line 381: / Line 381: @@
-          <p class="journal-content">Protein expression optimization experiments were performed on LbpA to assay protein production levels. However, cell growth seems to cease when LbpA expression is induced.</p>
+          <p class="journal-content">Protein expression optimization experiments were performed on LbpA to assay protein production levels. However, cell growth seems to cease when <i>LbpA</i> expression is induced.</p>
           </div>
@@ Line 388: / Line 388: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">1/7: LbpA + pQE80-L Overnight Cultures</span>
+             <span class="box-title">1/7: <i>LbpA</i> + pQE80-L Overnight Cultures</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-1"></span>
                  <div id="collapsiblew5-1" class="collapse box-content">
@@ Line 394: / Line 394: @@
                    <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#onc-modal" class="journal-protocol"> Overnight Cultures </a></p>
                <p><b>Results:</b> N/A </p>
-<p><b>Next Steps:</b> The overnight cultures will be used to set up day cultures tomorrow which will be used to test expression of the LbpA. </p>
+<p><b>Next Steps:</b> The overnight cultures will be used to set up day cultures tomorrow which will be used to test expression of LbpA. </p>
 </div>
          </div>
@@ Line 402: / Line 402: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">2/7: Optimization of LbpA Expression</span>
+             <span class="box-title">2/7: Optimization of <i>LbpA</i> Expression</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-2"></span>
                  <div id="collapsiblew5-2" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To set up day cultures and induce expression of LbpA using different concentrations of IPTG and check levels of protein expression via SDS-PAGE.</p>
+                   <p><b>Aim of experiment:</b> To set up day cultures and induce expression of <i>LbpA</i> using different concentrations of IPTG and check levels of protein production via SDS-PAGE.</p>
                    <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Optimization </a> </p>
                <p><b>Results:</b> N/A</p>
@@ Line 416: / Line 416: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">3/7: Continuation of Optimization of LbpA Expression</span>
+             <span class="box-title">3/7: Continuation of Optimization of <i>LbpA</i> Expression</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-3"></span>
                  <div id="collapsiblew5-3" class="collapse box-content">
@@ Line 443: / Line 443: @@
-          <p class="journal-content">Further protein expression optimization experiments were set up, this time using a range of IPTG concentrations to induce expression of LbpA.</p>
+          <p class="journal-content">Further protein expression optimization experiments were set up, this time using a range of IPTG concentrations to induce expression of <i>LbpA</i>.</p>
           </div>
@@ Line 450: / Line 450: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">7/7: Further Optimization of LbpA Expression</span>
+             <span class="box-title">7/7: Further Optimization of <i>LbpA</i> Expression</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-1"></span>
                  <div id="collapsiblew6-1" class="collapse box-content">
                    <p><b>Aim of experiment:</b> To set up further tests to optimize expression of LbpA using different concentrations of IPTG.</p>
-                   <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Optimization </a> 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. </p>
+                   <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Optimization </a> Note: An SDS gel was not ran, instead the OD<sub>600</sub> 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. </p>
                <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure5-modal" class="journal-protocol"> Figure 5 </a> </p>
-<p><b>Next Steps:</b> 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. </p>
+<p><b>Next Steps:</b> 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 <i>LbpA</i> expression has been induced. </p>
 </div>
          </div>
@@ Line 476: / Line 476: @@
-          <p class="journal-content">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.  </p>
+          <p class="journal-content">Protein expression optimization experiments were continued this week. A growth curve assay was carried out to assess the effect of <i>LbpA</i> expression over a longer period of time.  </p>
           </div>
@@ Line 486: / Line 486: @@
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew7-1"></span>
                  <div id="collapsiblew7-1" class="collapse box-content">
-                   <p><b>Aim of experiment:</b> To induce expression of LbpA using different concentration of IPTG and allow cells to grow for 16 hours in order to monitor cell growth.</p>
+                   <p><b>Aim of experiment:</b> To induce expression of <i>LbpA</i> using different concentration of IPTG and allow cells to grow for 16 hours in order to monitor cell growth.</p>
                    <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#platereader-modal" class="journal-protocol"> Growth Curve Assay </a></p>
                <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure6-modal" class="journal-protocol"> Figure 6 </a> </p>
-<p><b>Next Steps:</b> 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. </p>
+<p><b>Next Steps:</b> 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 <i>LbpA</i> expression has been induced. </p>
 </div>
          </div>
@@ Line 579: / Line 579: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">27/7: LbpA Overnight Cultures</span>
+             <span class="box-title">27/7: Overnight Cultures</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew9-1"></span>
                  <div id="collapsiblew9-1" class="collapse box-content">
@@ Line 593: / Line 593: @@
         <div class="border-day">
          <div class="box">
-             <span class="box-title">28/7: LbpA Expression Assay</span>
+             <span class="box-title">28/7: <i>LbpA</i> Expression Assay</span>
                <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew9-2"></span>
                  <div id="collapsiblew9-2" class="collapse box-content">
@@ Line 599: / Line 599: @@
                    <p><b>Protocols Used: </b> <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> Protein Expression Test </a> 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. </p>
                <p><b>Results:</b> <a data-toggle="modal" data-target="#lbpa-figure8-modal" class="journal-protocol"> Figure 8 </a>   </p>
-<p><b>Next Steps:</b> A 3L culture will be set up on Thursday under the same conditions as the overnight cultures in order to try and purify the LbpA.</p>
+<p><b>Next Steps:</b> A 3L culture will be set up on Thursday under the same conditions as the overnight cultures in order to try and purify LbpA.</p>
 </div>
          </div>

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