Team:Dundee/CGCraigon

CSI: DUNDEE

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Nasal Mucus

Week Beginning 15/6/2015

Summary

Produced a purified plasmid of pIDT-OBP2A in order to use it as template in PCR.

17/6: Transformation of pIDT-OBP2A in MC1610 E.coli strain

Aim of Experiment: To transform pIDT-OBP2A into MC1061 e.coli strain.

Protocols Used: Transformation

Results: N/A

Next Steps: The result of this transformation can be seen tomorrow. If successful then the next step will be to produce an overnight culture in preparation of subsequent plasmid purification.

18/6: Overnight culture of pIDT-OBP2A plasmid in MC1061

Aim of experiment: To produce an overnight culture of pIDT-OBP2A from positive colonies of MC1061 from the transformation done on the 17/6.

Protocols Used: Overnight Culture

Results: N/A

Next Steps: If the overnight culture successfully grows then the next stage of experimentation will be to produce a plasmid purification of pIDT-OBP2A from that overnight culture.

19/6: Plasmid Purification of pIDT-OBP2A from the overnight culture

Aim of experiment: The overnight culture produced on the 18/6 will now undergo a plasmid purification in order to obtain the pIDT-OBP2A plasmid.

Protocols Used: Plasmid Purification

Results: The plasmid purification yielded a concentration of 305.53 ng/ul.

Next Steps: Assuming the plasmid purification has been successful, then the next stage of experimentation will be to run a PCR using this purified pIDT-OBP2A plasmid as the template for subsequent amplification.

Week Beginning 22/6/2015

Summary

Removal of the signaling peptide from OBP2A and insertion of OBP2A into the biobrick vector pSB1C3.

22/6: PCR of pIDT-OBP2A using primers designed for the removal of the signal peptide

Aim of Experiment: To perform a PCR of the purified pIDT-OBP2A plasmid using a set of primers that have been designed to remove the signaling peptide at the start of the OBP2A gene, whilst leaving the rest of the gene intact.

Protocols Used: PCR

Results: N/A

Next Steps: If the PCR is successful, then the next step will be to gel extract OBP2A from a the gel corresponding to the size of the OBP2A gene fragment.

23/6: Gel extraction of OBP2A from the PCR performed on the 22/6

Aim of experiment: To perform a gel extraction of OBP2A from a PCR reaction that was performed on the 22/6. Once gel extraction has been performed a subsequent gel will be done to determine if the gel extraction had been successful.

Protocols Used: Gel Extraction

Results: Figure 1 Figure 2

Next Steps: To perform a restriction of OBP2A in preparation for ligation.

24/6: Restriction, ligation and transformation of OBP2A into pSB1C3

Aim of experiment: To perform a restriction, ligation and subsequently a transformation of of the gel extracted OBP2A into the JM110 E.coli strain.

Protocols Used: Restriction Digest : Ligation : Transformation

Results: N/A

Next Steps: If the transformation is successful and positive colonies form then these colonies will then be grown overnight in preparation for plasmid purification.

25/6: Re-ligation and transformation of OBP2A into pSB1C3

Aim of experiment: The transformation performed on the 24/6 failed and as a result OBP2A will now be re-ligated into the biobrick vector pSB1C3 and subsequently transformed into the JM110 E.coli strain.

Protocols Used: Ligation: Transformation

Results: Figure 3

Next Steps:If the transformation is successful and positive colonies form then these colonies will then be grown overnight in preparation for plasmid purification.

26/6: Overnight cultures of pSB1C3-OBP2A

Aim of experiment: To produce overnight cultures of pSB1C3-OBP2A from a positive colony from the transformation done on the 25/6.

Protocols Used: Overnight Cultures

Results: N/A

Next Steps: Produce plasmid purifications from the overnight cultures of pSB1C3-OBP2A in preparation for presequence digest.

27/6: Plasmid purification and presequence digest of pSB1C3-OBP2A

Aim of experiment: To perform a plasmid purification of the overnight cultures of pSB1C3-OBP2A from the 26/6. Then to subsequently perform a presequence digest of those plasmid purification.

Protocols Used: Plasmid Purification Presequence digest

Results: Figure 4

Sample	Concentration(ng/ul)
1	308.74
2	342.3
3	369.90
4	321.97
5	460.78
6	261.61

Next Steps: It was concluded from the results of these experiments that sample 5 of pSB1C3-OBP2A will be sent for sequencing.

Week Beginning 29/6/2015

Summary

Positive Sequencing of pSB1C3-OBP2A and subsequent plasmid purification of pSB1C3-OBP2A.

29/6: Plasmid purified pSB1C3-OBP2A is sent for sequencing

Aim of Experiment: Sample 5 of the plasmid purified pSB1C3-OBP2A that was done on the 24/6 is sent for sequencing to determine if OBP2A has successfully ligated into pSB1C3.

Protocols Used: N/A

Results: N/A

Next Steps: If sequencing comes back positive, then the next step will be to clone OBP2A into the two vectors of the bacterial two hybrid system.

30/6: Overnight culture of pSB1C3-OBP2A from the sequenced colony

Aim of Experiment: Sequencing came back positive for pSB1C3-OBP2A. So an overnight culture of pSB1C3-OBP2A will be done in preparation for plasmid purification.

Protocols Used:Overnight Cultures

Results: N/A

Next Steps: Once the overnight culture has been given ample time to grow, the culture will then undergo a plasmid purification.

1/7: Plasmid Purification of pSB1C3-OBP2A

Aim of Experiment: To produce a plasmid purification of the sequenced pSB1C3-OBP2A from the overnight culture set up on the 30/6.

Protocols Used:Plasmid Purification

Results: The plasmid purified pSB1C3-OBP2A produced a concentration of 448.83 ng/ul.

Next Steps: The next stage of experimentation is to use the plasmid purification of pSB1C3-OBP2A as the template for PCR using primers that are designed to separate the gene for use in the two hybrid system.

Week Beginning 12/7/2015

Summary

Ran PCR of OBP2A using primers for insertion of OBP2A into pUT18 and pT25.

17/7: PCR of OBP2A for insertion into bacterial two hybrid vectors: pUT18 and pT25

Aim of Experiment: To perform a PCR of OBP2A for its insertion into the bacterial two hybrid vectors; pUT18 and pT25.

Protocols Used: PCR

Results: N/A

Next Steps:The next stage of experimentation will be to gel extract and then subsequently restrict the amplified gene fragments of OBP2A.

Week Beginning 20/7/2015

Summary

Attempted insertion of OBP2A into the vectors for the bacterial two hybrid system; pUT18 and pT25.

20/7: Gel extraction and restriction of OBP2A from the PCR done on the 17/7

Aim of Experiment: To perform a gel extraction and then subsequently a restriction of OBP2A from the PCR produced on the 17/7 using primers designed for the insertion of OBP2A into the vectors for the bacterial two hybrid system, pUT18 and pT25.

Protocols Used: Gel Extraction Restriction Digest

Results: N/A

Next Steps: If the gel extraction and subsequent restriction is successful, then the next step will be to ligate the restricted OBP2A into the bacterial two hybrid vector, pUT18 and pT25.

21/7: Ligation and transformation of OBP2A into pUT18 and pT25.

Aim of Experiment: To perform a ligation and then subsequently a transformation of OBP2A into the vectors for the bacterial two hybrid system, pUT18 and pT25. Whilst using JM110 E.coli cells as a chassis for transformation.

Protocols Used: Ligation Transformation

Results: N/A

Next Steps: The results from the transformation will be seen tomorrow. If successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight colonies in preparation for purification and sequencing.

22/7: Re-ligation and transformation of OBP2A into pUT18 and PT25.

Aim of Experiment: To perform a re-ligation and transformation of OBP2A into pUT18 and pT25 - since the transformation performed on the 21/7 failed.

Protocols Used: ligation Transformation

Results: N/A

Next Steps: If transformation is successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight colonies in preparation for purification and sequencing.

23/7: Production of overnight cultures of pUT18-OBP2A.

Aim of Experiment: To produce overnight cultures of pUT18-OBP2A. Unfortunately no positive colonies were produced from the transformation of OBP2A into pT25.

Protocols Used: Overnight Culture

Results: N/A

Next Steps: Once the cells have been given ample time to grow, they will undergo plasmid purification for subsequent sequencing.

24/7: Plasmid purification and subsequent presequence digest of pUT18-OBP2A

Aim of Experiment: To perform a plasmid purification of the overnight cultures of pUT18-OBP2A and then a subsequent presequence digest.

Protocols Used: Plasmid Purification Presequence digest

Results: Figure 5

Next Steps: If sequencing comes back positive for pUT18-OBP2A then this can now be implemented into the bacterial two hybrid system. Another attempt at cloning OBP2A into pT25 will be done next week.

Week Beginning 27/7/2015

Summary

Successful insertion of the two subunits of OBP2A into the vectors pUT18 and pT25.

27/7: PCR and gel extraction of OBP2A for preparation of insertion into pT25.

Aim of Experiment: To perform a PCR and subsequent gel extraction of OBP2A for its insertion into pT25.

Protocols Used: PCR

Results: Figure 6

Next Steps: The next stage of experimentation will be to restrict OBP2A for subsequent ligation into pT25.

28/7: Restriction of OBP2A for pT25 and positive sequencing of pUT18-OBP2A

Aim of Experiment: To perform a restriction of OBP2A for subsequent ligation into PT25. Also, sequencing came back positive for pUT18-OBP2A, so pUT18-OBP2A can now be implemented in the bacterial two hybrid system.

Protocols Used: Restriction Digestion

Results: N/A

Next Steps: The next stage of experimentation will be to ligate OBP2A into the pT25 vector.

30/7: Ligation and transformation of OBP2A into pT25.

Aim of Experiment: To perform a ligation and transformation of OBP2A into the bacterial two hybrid vector pT25, so that the interaction between the two constituents of OBP2A can be observed using the bacterial two hybrid system.

Protocols Used: ligationTransformation

Results: N/A

Next Steps: If the transformation is successful and positive colonies form on the antibiotic plates, then the next step will be to grow overnight cultures of those colonies in preparation for purification and sequencing.

31/7: Overnight cultures of pT25-OBP2A from positive colonies.

Aim of Experiment: To produce overnight cultures of pT25-OBP2A from the transformation done on the 30/7.

Protocols Used: Overnight Culture

Results: N/A

Next Steps: Once the cells have grown overnight they will undergo plasmid purification for subsequent sequencing.

1/8: Plasmid purification and subsequent presequence digest of pT25-OBP2A

Aim of Experiment: To perform a plasmid purification of pT25-OBP2A from the overnight cultures done on the 31/7 and then a subsequent presequence digest.

Protocols Used: Plasmid PurificationPresequence digest

Results: Figure 7

Purified Plasmid Sample	Concentration(ng/ul)
1	50.37
2	42.36
3	37.13
4	14.87
5	80.02
6	44.34
7	22.83

Next Steps: If sequencing comes back positive for pT25-OBP2A then this can now be implemented into the bacterial two hybrid system. The results from the sequencing should arrive early on the 3/8.

Week Beginning 3/8/2015

Summary

Positive sequencing of pT25-OBP2A, subsequent transformation into the bacterial two hybrid system and plating onto MacConkey agar.

3/8: Transformation of pUT18-OBP2A into BTH101 E.coli strain.

Aim of Experiment: To perform a transformation of pUT18-OBP2A into BTH101, so that a subsequent transformation of pT25-OBP2A into the same chassis can occur as both plasmids are required to reside in the same chassis so that their interaction can be observed.

Protocols Used: Transformation

Results: N/A

Next Steps: If the transformation is successful, then the next stage of experimentation will be to re-transform this culture of cells using the pT25-OBP2A plasmid.

4/8:Day culture and transformation of BTH101 containing pUT18-OBP2A with pT25OBP2A

Aim of Experiment: To perform a day culture and then a subsequent transformation of the BTH101 chassis containing pUT18-OBP2A from the transformation performed on 3/8.

Protocols Used: Transformation

Results: N/A

Next Steps: If the transformation is successful then the next stage of experimentation will be to observe the protein interaction of the two subunits of OBP2A using MacConkey agar plates.

5/8:Day culture and plating of positive colonies from transformation onto MacConkey agar.

Aim of Experiment: To perform a day culture and then subsequent plating of positive colonies from the transformation performed on the 4/8. Positive colonies from this transformation contain both vectors of the bacterial two hybrid system along with the two subunits of OBP2A and so now the interaction can be observed. The day culture will be plated onto MacConkey agar which once the cells have been given enough time to grow will give a visual demonstration of the protein interaction through a color change.

Protocols Used: Transformation

Results: N/A

Next Steps: It has been decided that whilst the E.coli strain BTH101 that is being used in this experiment is useful for visualizing protein interaction, it is very unstable. A better E.coli strain called MG1655(Δcya) will be used instead as it is much more stable. This means that tomorrow overnights will be prepared of MG1655(Δcya)for transformation with pUT18-OBP2A and then subsequently pT25-OBP2A.

6/8: Overnight cultures of MG1655(Δcya)

Aim of Experiment: To perform an overnight culture of MG1655(Δcya)E.coli in preparation for transformation with pUT18-OBP2A and then pT25-OBP2A.

Protocols Used: Overnight Culture

Results: N/A

Next Steps: Once the overnight culture has been give sufficient time to grow these cells will then be transformed with pUT18-OBP2A and then subsequently re-transformed with pT25-OBP2A.

7/8: Results of MacConkey agar growth and transformation of MG1655(Δcya)with pUT18-OBP2A

Aim of Experiment: To perform a transformation of MG1655(Δcya)with pUT18-OBP2A. Also the results of plating of our bacterial two hybrid interaction onto MacConkey agar has produced promising results as the colonies appear to be taking up a small amount of red dye - an indicator of protein interaction. This suggests that the two separated parts of OBP2A may in fact be interacting in vivo. This interaction will need to be quantified through the use of the β-galactosidase assay.

Protocols Used: Transformation

Results: N/A

Next Steps: If the transformation of MG1655(Δcya) with pUT18-OBP2A is successful then the next step of experimentation will be re-transform positive colonies with pT25-OBP2A.

8/8: Transformation of MG1655(Δcya) containing pUT18-OBP2A with pT25-OBP2A

Aim of Experiment: To perform a transformation of MG1655(Δcya) containing pUT18-OBP2A with pT25-OBP2A. Positive colonies were produced from the transformation done on the 7/7 and so as a result a day culture was produced and subsequently re-transformed with pT25-OBP2A.

Protocols Used: Transformation

Results: N/A

Next Steps: If the transformation of MG1655(Δcya) with pT25-OBP2A is successful then the next step of experimentation will be to undertake the β-galactosidase assay

Week Beginning 10/8/2015

Summary

First attempt at determination of the protein interaction of the OBP2A subunits using the β-galactosidase assay.

10/8: Plating of Controls in preparation of the β-galactosidase assay

Aim of Experiment: To plate control interactions that are used in the β-galactosidase assay to aid in the interpretation of the results form the assay. This first assay attempt will consist of one positive control, one negative control and one regulatory control.

Protocols Used: N/A

Results: N/A

Next Steps: The next stage of experimentation will be to perform overnight cultures from positive colonies in preparation for the β-galactosidase assay.

11/8: Overnight cultures of controls and OBP2A in preparation for the β-galactosidase assay.

Aim of Experiment: To prepare overnight cultures of positive colonies of MG1655(Δcya) containing pUT18-OBP2A and pT25-OBP2A, along with the controls in preparation for the β-galactosidase assay.

Protocols Used: Overnight Cultures

Results: N/A

Next Steps: Once the cells have been given sufficient time to grow then the next stage of experimentation will be to inoculate those colonies into fresh liquid broth until they reach an O.D of 0.4 before being pellet and frozen for the assay.

12/8: Inoculation and preparation of cultures for the β-galactosidase assay.

Aim of Experiment: To prepare cultures for use in the β-galactosidase assay, this requires a small subsample of each overnight to be inoculated into fresh LB, two done for each colony. Then left to grow until the O.D of each culture is between 0.3-0.5. Once an O.D of 0.3-0.5 has been reached then 3 replicas of 1 ml samples are taken from each culture before being spun down, the supernatant then subsequently removed and the pellet frozen.

Protocols Used: Assay Preparation

Results: N/A

Next Steps: These samples are now ready for the β-galactosidase assay.

13/8: First attempt at the determination of the protein interaction of the two OBP2A subunits using the β-galactosidase assay

Aim of Experiment: To determine the millers activity of the two interacting subunits of OBP2A in vivo as a basis of the level of interaction between them. This was done using the β-galactosidase assay.

Protocols Used: β-galactosidase assay

Results: Figure 8

Next Steps: As it was the first attempt at a β-galactosidase assay, subsequent assays will be performed utilizing more controls.

14/8: Preparation of overnight cultures of controls and samples in preparation of performing a second β-galactosidase assay

Aim of Experiment: To perform overnight cultures of the controls and samples that will be necessary for doing this second β-galactosidase assay. This assay will include two positive controls which are known to have a high level of interaction - these are NarG-NarJ and Zip positive controls. There will also be two negative controls, an empty MG1655(Δcya) chassis and pUT18-OBP2A with NarJ. Finally there is one regulatory control: which contains two empty vectors of pUT18 and pT25.

Protocols Used:Overnight Cultures

Results: N/A

Next Steps: The next stage of experimentation will be to prepare samples for use in the β-galactosidase assay.

Week Beginning 17/8/2015

Summary

Second attempt at determining the protein interactions of the two OBP2A subunits using the β-galactosidase assay and subsequent cloning of OBP2A into the bacterial two hybrid vectors in switched order.

17/8: Preparation of the controls and samples for use in the second attempt of the β-galactosidase assay

Aim of Experiment: To prepare cultures for use in the β-galactosidase assay, this requires a small subsample of each overnight to be inoculated into fresh LB, two done for each colony. Then left to grow until the O.D of each culture is between 0.3-0.5. Once an O.D of 0.3-0.5 has been reached then 3 replicas of 1 ml samples are taken from each culture before being spun down, the supernatant then subsequently removed and the pellet frozen.

Protocols Used: Assay Preparation

Results: N/A

Next Steps: The next stage of experimentation will be to perform the second β-galactosidase assay on the interaction between the two subunits of OBP2A

18/8: Second attempt at determination of the protein interactions of the OBP2A subunits using the β-galactosidase assay

Aim of Experiment: To perform a second attempt at the β-galactosidase assay that will aid in the determination of the level of interaction that the two separated subunits of OBP2A are inflicting on one another.

Protocols Used: β-galactosidase assay

Results: Figure 10

Next Steps: The next stage of experimentation will be to switch the positioning of the two subunits of OBP2A in pUT18 and pT25 by having the smaller subunit in pUT18 and the large subunit in pT25. Along with this another β-galactosidase assay will be performed however this time it will be done under anaerobic conditions to see if that has any appreciable effect on the interaction of the two subunits of OBP2A.

20/8: PCR of OBP2A in preparation of pUT18 and pT25 insertion

Aim of Experiment: To Perform a PCR of OBP2A using primers for pU1T18 and pT25 insertion which have been designed to place the smaller OBP2Asubunit into pUT18 and the larger subunit into pT25.

Protocols Used: PCR

Results: N/A

Next Steps: If PCR is successful then the next stage of experimentation will be to perform a gel extraction and restriction in perpetration for ligation and transformation of OBP2A into pUT18 and pT25.

21/8: Second attempt at a PCR of OBP2A in preparation of pUT18 and pT25 insertion

Aim of Experiment: The PCR performed on the 20/8 failed as no bands were found to be present on the gel during gel viewing. As a result of this a subsequent PCR was done today.

Protocols Used: PCR

Results: N/A

Next Steps: If PCR is successful then the next stage in experimentation will be to perform a gel extraction and restriction in perpetration for ligation and transformation of OBP2A in pUT18 and pT25.

22/8: Second attempt at PCR of OBP2A for insertion into pUT18 and pT25 failed

Aim of Experiment: A second attempt at preparing OBP2A for insertion into the two vectors of the bacterial two hybrid system(pUT18 and pT25) has failed! As a result this will be left until next week, so that the rest of the week can be dedicated to preparing and undertaking another β-galactosidase assay, that will be done under anaerobic conditions.

Protocols Used: N/A

Results: N/A

Next Steps: The next stage of experimentation is to prepare for a anaerobic β-galactosidase assay of OBP2A in the bacterial two hybrid vectors; pUT18 and pT25.

Week Beginning 24/8/2015

Summary

Performed an anaerobic β-galactosidase assay in order to determine the level of interaction between the two subunits of OBP2A.

24/8: Preparation of samples for the anaerobic β-galactosidase assay of OBP2A in pUT18 and pT25

Aim of Experiment: To prepare the samples and controls of OBP2A for the anaerobic β-galactosidase assay, so they can be frozen overnight in preparation for the assay itself.

Protocols Used: Assay Preparation

Results:

Next Steps: The next stage of experimentation will be to perform the β-galactosidase assay itself.

25/8: Performed an anaerobic β-galactosidase assay in order to determine the level of interaction of OBP2A in the bacterial two hybrid vectors: pUT18 and pT25.

Aim of Experiment: Performed an anaerobic β-galactosidase assay of OBP2A in the bacterial two hybrid vectors: pUT18 and pT25. This is to determine the level of interaction that the smaller subunit of OBP2A has with its larger subunit in vivo. This particular assay has two positive controls, two negative controls and one regulatory control.

Protocols Used: β-galactosidase assay

Results: Figure 12

Next Steps: The results have shown that the two subunits of OBP2A in fact don't seem to be interacting in vivo - a possible reason for this might be aggregation of the two subunits preventing interaction. The next stage of experimentation will be to test out the interaction when the subunit are switch in relation to heir positions on the vectors: pUT18 and pT25.

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Figure 1: Amplification of OBP2A for insertion into pSB1C3 vector . This is the PCR product from the amplification of OBP2A from the IDT plasmid using primers for cloning into pSB1C3. The gel indicates OBP2A has been amplified successfully since the observed band corresponds to the expected size of 500 bp. The concentration of the gel extracted OBP2A is 335.53 ng/ul.

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Figure 2: A subsequent gel was run of the amplified OBP2A after it was first excised from the gel in figure 1 - this was to determine if the gel extraction was successful. The gel indicates that OBP2A has been extracted from the gel successfully, since the observed band corresponds to the expected size of 500 bp. This can now be restricted in preparation for ligation into pSB1C3.

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Figure 3: A gel analysis of my restriction digest from the 24/6. This gel analysis was done to determine if the reason for the failed transformation was an improperly digested OBP2A. This gel indicates that the restricted product hasn't been lost as the OBP2A gene fragment can be seen as a distinct band at 500 bp. A re-ligation of OBP2A into the pSB1C3 backbone will now occur.

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Figure 4: Presequence Digest of pSB1C3-OBP2A. All seven plasmid purifications of the overnight cultures done on the 26/6 were digested to see if they contained the OBP2A insert. It is clear from the gel that all but sample 3 contain the insert for OBP2A as they all have faint bands at 500 bp's which corresponds to the size of OBP2A. It also appears that sample 5 contains the highest concentration, so that will be the sample that is sent for sequencing.

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Figure 5: Presequence digest of pUT18-OBP2A to determine if any samples contain the OBP2A insert. It can be seen from this gel that of the 5 samples that were digested only two are show to have the OBP2A insert, therefore sample 2 will be sent for sequencing.

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Figure 6: Amplification of OBP2A for insertion into pT25 vector . This is the PCR product from the amplification of OBP2A for cloning into pT25. The gel indicates OBP2A has been amplified successfully since the observed band corresponds to the expected size of 100 bp.

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Figure 7: Presequence Digest of pT25-OBP2A, in order to determine if any of the samples contain the OBP2A insert. It can be seen from this gel that samples 5, 6 and 7 may have inserts in them as they each have two distinct bands, one of which corresponds to the 100 bp size for the OBP2A for pT25. As a result all three samples will be sent for sequencing.

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Figure 8: A graph of the results from the first attempt at a β-galactosidase assay that aimed to characterize the interaction between the two separated parts of OBP2A. As it can be seen that the positive result has failed(far left) not much can be inferred from the interaction of the two parts of OBP2A. However it looks like the two subunits of OBP2A may not be interacting. As this was a first attempt, subsequent assays will be performed in order to determine if in fact the two separated parts of OBP2A aren't interacting in vivo - like these results suggests.

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Figure 9: A graph of the results from the second attempt at a β-galactosidase assay that aimed to characterize the interaction between the two separated parts of OBP2A. It can also be inferred from this graph that the two parts of OBP2A don't seem to be interacting as it has a low millers activity when compared to the controls. Further assays will be performed in order to determine if the two separated parts of OBP2A aren't interacting in vivo like these results suggests.

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Figure 11: A graph of the results from the anaerobic β-galactosidase assay aimed to characterize the interaction between the two separated parts of OBP2A. The graph seems to suggest that the two subunits of OBP2A may not be interacting in vivo. This can be inferred from the fact that the millers activity of the OBP2A sample lies lower than the negative controls from within the experiment. As a result of this information, one more assay will be prepared in which the two subunits will be switched with regards to the bacterial two hybrid vectors to eliminate any possibility of this being a factor effecting the interaction between the two subunits.

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Overnight Culture Protocol

1. Agar plates with required antibiotic were taken out of 4^oC to warm to room temperature.
2. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
3. 5ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.
4. Tubes were incubated at 37^oC in a rotary incubator at 200rpm over night.

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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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Miniprep Protocol

1. 5ml bacterial overnight culture was pelleted by stepwise centrifugation in 1.5ml microcentrifuge tubes at 13300rpm for 3 minutes at room temperature.
2. Pelleted bacterial cells were resuspended in 250µl of Buffer P1.
3. 250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution became 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 13300rpm in a microcentrifuge.
6. The supernatant was applied to a QIAprep spin column by pipetting.
7. 500µl of Buffer PB was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.
8. 750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was 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 30µl of H₂O to the QIAprep spin column and centrifuging for 1 minute.

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Gel Extraction Protocol

1. The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel.
2. The gel slice was transferred into a microcentrifuge tube. 700µl of Buffer QG was added.
3. The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel.
4. After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow.
5. 230µl of isopropanol was added to the sample and mixed.
6. A QIAquick spin column was placed in a 2ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
7. 500µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
8. 750µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was 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 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute.

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

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Assay Preparation

1. Take 3 independent colonies from each plate of your controls and samples and place each colony into 5 ml of LB with the appropriate antibiotics.
2. Leave to grow overnight at 30°C.
3. Take 50 μl of each overnight and inoculate 5 ml of fresh LB containing the appropriate antibiotics. Do this twice for every independent colony.
4. Allow these to grow at 30°C shaker until the O.D of each inoculate is between 0.3-0.5
5. Once the O.D is between 0.3-0.5 take 3 x 1 ml samples of each culture and place into separate Eppendorf tubes.
6. Take a further 1 ml and place into a cuvette and subsequently measure the O.D at 600 nm marking down the O.D for each replica.
7. Spin down the 1 ml cultures of each replica at 13,000 rpm for 3 minutes.
8. Remove th supernatant from each 1 ml culture and freeze cell pellet for later use in the β-galactosidase Assay.

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β-galactosidase Assay

1. Resuspend cell pellet in 800ul of buffer 2 (100 mM dibasic sodium phosphate (Na2HPO4); 20 mM KCl ; 2 mM MgSO4 ;0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide) ; 0.4 mg/mL sodiumdeoxycholate) and subsequntly add 5.4 μL/mL beta-mercaptoethanol.
2. Add 50 ul of SDS (0.1%) to each sample.
3. Add 50 ul of Chloroform to each sample and vortex for 30 seconds.
4. Make substrate solution (60 mM Na2HPO4; 40 mM NaH2PO4; 1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG); 2.7 μL/mL β-mercaptoethanol)
5. Add 200 ul of substrate solution to each sample marking down the time it was added in relation to the first sample.
6. Wait for an observable color change and mark down the time that the color change occurred.
7. Add 600 ul of NaCO3(1M) once the color change has occurred.
8. Spin down the samples at 13,000 rpm for 3 minutes and place the supernatant into a cuvette
9. Measure the O.D of each sample at 420 nm

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