Team:Dundee/lucylabjournal

LABJOURNAL

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

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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 gene from N.meningitidis template.

Protocols Used: Click here to see our PCR protocol.

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 JM110 cells.

3/6: Transformations of LpbA + pSB1C3 into JM110

Aim of experiment: To transform the pSB1C3 + LbpA ligations into JM110 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

Week summary

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8/6: Amplification, Restriction Digest and Ligation 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

9/6: Restriction Digests and Ligations of LbpA into 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

Week summary

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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

Week summary

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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

Results: N/A

Next Steps: The ligations will be transformed into the M15pREP4 strain of E.coli.

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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.

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Restriction Digest Protocol

Restriction digests were set up according to the table below. The appropriate reaction and restriction enzymes that were used is stated in the Journal 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 its buffer was added at the 2 hour and 2.5 hour mark.

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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 Journal 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.

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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.

Cells were plated on agar plates with appropriate antibiotic.

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Figure 1: LbpA PCR. 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.

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Figure 2: LbpA PCR. 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.

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Figure 3: LbpA PCR for Cloning into pQE80-L. 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.

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