Team:Dundee/cgcraigon
Watch Our Introduction Video
Summary
Produced a plasmid preparation of pIDT-OBP2A in order to use it as template in PCR.
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.
Aim of experiment: To produce an overnight culture of pIDT-OBP2A from positive colonies of MC1061 E.coli strain from the transformation done on the 17/6.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: If the overnight culture successfully grows then the next stage of experimentation will be to produce a plasmid preparation of pIDT-OBP2A from this overnight culture.
Aim of experiment: The overnight culture produced yesterday will now undergo a plasmid preparation in order to obtain the pIDT-OBP2A plasmid.
Protocols Used: Miniprep
Results: The plasmid preperation yeilded a concentration of 305.53 ng/ul.
Next Steps: To run a PCR using the purified pIDT-OBP2A plasmid as a template for amplification.
Summary
Removal of the signaling peptide from OBP2A and insertion of OBP2A into the biobrick vector pSB1C3.
Aim of Experiment: To perform a PCR on the plasmid preparation of pIDT-OBP2A produced last week, using primers that have been designed in a way as to remove the signalling peptide at the start of the OBP2A gene.
Protocols Used: PCR
Results: N/A
Next Steps: The next step will be to run the PCR product on a gel and perform a gel extraction on the part of the gel corresponding to the size of the OBP2A gene fragment.
Aim of experiment: To perform a gel extraction of OBP2A from a PCR reaction mixture 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 has been successful.
Protocols Used: Gel extraction
Results: As it can be seen from the gel image, a band is present corresponding to the size of the OBP2A gene fragment.
Next Steps: To perform a restriction of OBP2A in preparation for ligation.
Aim of experiment: To firstly restrict the gel extracted OBP2A. Secondly, to then ligate the restricted OBP2A into the biobrick vector pSB1C3. Third and finally to transform this plasmid 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 on the agar plate then these colonies will then be grown overnight in preparation for Miniprep.
Aim of experiment: The transformation performed on the 24/6 failed and as a result OBP2A will again ligated into the biobrick vector pSB1C3 and then transformed into JM110 E.coli strain.
Protocols Used: Ligation: Transformation
Results: N/A
Next Steps:If the transformation is successful and positive colonies form on the agar plate then these colonies will then be grown overnight in preparation for Miniprep.
Aim of experiment: To produce overnight cultures of pSB1C3-OBP2A from positive colonies of JM110 E.coli strain from the transformation done on the 25/6.
Protocols Used: Overnight Cultures
Results: N/A
Next Steps: Produce a plasmid preparation of pSB1C3-OBP2A in preparation for pre-sequence digest.
Aim of experiment: To perform a plasmid preparation of overnight cultures of pSB1C3-OBP2A from positive colonies of JM110 E.coli strain from the transformation done on the 25/6. Then to subsequently perform a pre-sequence digest of the plasmid preparation.
Protocols Used: Overnight Cultures
Results: Insert table values!
Next Steps: Looking at the pre-sequence digest it has been decided that sample 5 will be sent for sequencing.
Summary
Correct insertion of OBP2A into pSB1C3 determined through sequencing.
Aim of Experiment: Sent sample 5 of the plasmid preparation that was done on the 24/6 for sequencing to determine if OBP2A has successfully ligated into pSB1C3.
Protocols Used:
Results: N/A
Next Steps: If sequencing comes back positive, then the next step will be to clone this gene insert into the two constituents of the two hybrid system.
Aim of Experiment: Sequencing came back positive for pSB1C3-OBP2A. Soan overnight culture of pSB1C3-OBP2A from the sequenced colony will be done overnight in preparation for plasmid preparation.
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 preparation.
Aim of Experiment: To produce a plasmid preparation of the sequenced pSB1C3-OBP2A from the overnight culture set up on the 30/6.
Protocols Used:Miniprep
Results: N/A
Insert plasmid concentration.Next Steps: the next step of experimentation is to use the plasmid preparation as a template for PCR using primer designed to separate the gene for use in the two hybrid system.
Summary
Ran PCR of OBP2A using primers for insertion of OBP2A into PUT18 and PT25.
Aim of Experiment: To PCR OBP2A for its insertion into the bacterial two hybris vectors; pUT18 and pT25.
Protocols Used: Transformation
Results: N/A
Next Steps:Tthe next stage of experimentation will be to gel extract and restrict the PCR gene fragments.
Summary
Attempted insertion of OBP2A into the vectors for the bacterial two hybrid system.
Aim of Experiment: To perform a gel extraction and then subsequently a restriction of OBP2A from the PCR produced on the 17/7 that used primers designed for the insertion ofOBP2A into the vectors for the bacterial two hybrid system, pUT18 and pT25.
Protocols Used: Gel extraction Restriction
Results: N/A
Next Steps: If the gel extraction and restriction is successful, then the next step will be to ligate these restrictions into the bacterial two hybrid vectors, 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 Transfromation
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.
Aim of Experiment: As the transformation from the 21/7 failed - no positive colonies grew on the antibiotic plates. A re-ligation and transformation of OBP2A into pUT18 and pT25 was done.
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.
Aim of Experiment: Although the transformation of OBP2A into pT25 was unsuccessful, the transformation of OBP2A into pUT18 did produce positive colonies. These colonies were grown overnight in preparation for Miniprep.
Protocols Used: Overnight Culture p>
Results: N/A
Next Steps: Once the cells have grown overnight they will undergo plasmid preparation for subsequent sequencing.
Aim of Experiment: The overnight cultures of pUT18-OBP2Aunderwent plasmid preparation and then a subsequent pre-sequence digest before being sent for sequencing.
Protocols Used: Miniprep Pre-sequence digest p>
Results: N/A
Next Steps: If sequencing comes back positive for pUT18-OBP2A then this can now be implemented into the bacterial two hybrid. Second attempt at cloning of OBP2A into pT25 will be done next week.
Summary
Successful insertion of OBP2A into both bacterial two hybrid vectors pUT18 and pT25.
Aim of Experiment: A restriction of OBP2A for PT25 was performed. Sequencing came back positive for pUT18-OBP2A, so pUT18-OBP2A can now be implemented in the bacterial two hybrid system.
Protocols Used: Restriction p>
Results: N/A
Next Steps: The next stage of experimentation will be to ligate OBP2A into the pT25 vector.
Aim of Experiment: As the restriction was done yesterday a ligation and transformation of OBP2A into the bacterial two hybrid vector pT25 was done today.
Protocols Used: ligationTransformation
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 cultures of those colonies in preparation for purification and sequencing.
Aim of Experiment: The transformation of OBP2A into pT25 produced positive colonies, so overnight cultures of these colonies were done today.
Protocols Used: Overnight Culture p>
Results: N/A
Next Steps: Once the cells have grown overnight they will undergo plasmid preparation for subsequent sequencing.
Aim of Experiment: The overnight cultures of pT25-OBP2Aunderwent plasmid preparation and then a subsequent pre-sequence digest before being sent for sequencing.
Protocols Used: Miniprep Pre-sequence digest p>
Results: N/A
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.
Summary
Aim of Experiment: In order to gauge the interaction between the two separate parts of my protein both pT25-OBP2A and pUT18-OBP2A must be transformed into the same cell. In this case the transformation is being done with the BTH101 E.coli strain.This is the first of two transformations.
Protocols Used: Trasformation
Results: N/A
Next Steps: If the transformation is successful, then the next stage of experimentation will be to retransform this culture of cells using the pT25-OBP2A plasmid.
Aim of Experiment: The transformation performed yesterday has produced positive colonies. These positive colonies were then grown throughout the day before being made competent and used in the transformation of pT25-OBP2A
Protocols Used: Transformation p>
Results: N/A
Next Steps: If the transformation is successful then the next stage of experimentation will be to observe the interaction of the two subunits in vivo using MacConkey agar plates.
Aim of Experiment: Positive colonies have formed on the agar plate of yesterday's transformation. Since this means that those positive colonies contain both bacterial two hybrid vectors then it is now possible to plate them on MacConkey agar as this will give us a visual demonstration of protein interaction.
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 protein interaction. A better E.coli strain called MG1655(-cya) will be used instead as it is far more stable. This means that tomorrow overnights will be prepared of MG1655(-cya)for transformation with pUT18 and pT25 containing the different subunits of OBP2A.
Aim of Experiment: Performed overnight of MG1655(-cya)E.coli in preparation for transformation with pUT18-OBP2A and pT25-OBP2A.
Protocols Used: Overnight Culture p>
Results: N/A
Next Steps: Once the overnight culture has been give the correct amount of time to grow these cells will be made competent and transformed with pUT18-OBP2A and pT25-OBP2A.
Aim of Experiment: Colonies of BTH101 that have formed on the MacConkey agar have grown to sufficient size that it is now possible to see that the colonies are uptaking a small amount of the red dye - which possibly suggests that the two separated parts of OBP2A are interacting. This will be shown through the use of the betagalactosidae assay. Cells of MG1655(-cya) from yesterdays overnight were firstly made competent then subsequently transformed with pUT18-OBP2A.
Protocols Used: Miniprep Pre-sequence digest p>
Results: N/A
Next Steps: If the transformation of MG1655(-cya) with pUT18-OBP2A is successful then the next step of experimentation will be retransform positive colonies with pT25-OBP2A.
Summary
Aim of Experiment: In order to perform the betagalactosidase assay several controls are implemented to allow for a concise interpretation of the results. This first assay attempt will consist of one positive control, one negative control and one intermediate control.
Protocols Used: Trasformation
Results: N/A
Next Steps: The next step will be to perform overnights in preparation of the betagalctosidase assay.
Aim of Experiment: Prepared overnights of positive colonies of MG1655(-cya) containing pUT18-OBP2A and pT25-OBP2A, along with the control colonies in preparation for the betagalactosidase assay.
Protocols Used: Overnight Cultures p>
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 free liquid broth until they reach an O.D of 0.4 before being pellet and frozen for the assay. This will be done tomorrow.
Aim of Experiment: A small subsample of each overnight was inoculated into fresh LB, there were two done for each colony. These were then left to grow until the O.D of each culture was between 0.3-0.5. Once there 3 replicas of 1ml were taken form each culture before being spun down, the supernatant removed and frozen.
Protocols Used: Transformation
Results: N/A
Next Steps: The samples are now ready for the betagalcatsidase assay which will be done tomorrow.
Aim of Experiment: To determine the millers activity of the subunits of OBP2A when interacting in vivo as a basis of the level of interaction between them. This was done using the betagalactosidase assay.
Protocols Used: Overnight Culture p>
Results: N/A
Next Steps: Since it isn't clear from the data that the subunits of OBP2A aren't interaction in vivo a further assay will be prepared using more controls.
Aim of Experiment: Colonies of BTH101 that have formed on the MacConkey agar have grown to sufficient size that it is now possible to see that the colonies are uptaking a small amount of the red dye - which possibly suggests that the two separated parts of OBP2A are interacting. This will be shown through the use of the betagalactosidae assay. Cells of MG1655(-cya) from yesterdays overnight were firstly made competent then subsequently transformed with pUT18-OBP2A.
Protocols Used: Miniprep Pre-sequence digest p>
Results: N/A
Next Steps: If the transformation of MG1655(-cya) with pUT18-OBP2A is successful then the next step of experimentation will be retransform positive colonies with pT25-OBP2A.
Summary
Aim of Experiment: In order to perform the betagalactosidase assay several controls are implemented to allow for a concise interpretation of the results. This first assay attempt will consist of one positive control, one negative control and one intermediate control.
Protocols Used: Trasformation
Results: N/A
Next Steps: The next step will be to perform overnights in preparation of the betagalctosidase assay.
Aim of Experiment: Prepared overnights of positive colonies of MG1655(-cya) containing pUT18-OBP2A and pT25-OBP2A, along with the control colonies in preparation for the betagalactosidase assay.
Protocols Used: Overnight Cultures p>
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 free liquid broth until they reach an O.D of 0.4 before being pellet and frozen for the assay. This will be done tomorrow.
Aim of Experiment: A small subsample of each overnight was inoculated into fresh LB, there were two done for each colony. These were then left to grow until the O.D of each culture was between 0.3-0.5. Once there 3 replicas of 1ml were taken form each culture before being spun down, the supernatant removed and frozen.
Protocols Used: Transformation
Results: N/A
Next Steps: The samples are now ready for the betagalcatsidase assay which will be done tomorrow.
Aim of Experiment: To determine the millers activity of the subunits of OBP2A when interacting in vivo as a basis of the level of interaction between them. This was done using the betagalactosidase assay.
Protocols Used: Overnight Culture p>
Results: N/A
Next Steps: Since it isn't clear from the data that the subunits of OBP2A aren't interaction in vivo a further assay will be prepared using more controls.
Aim of Experiment: Colonies of BTH101 that have formed on the MacConkey agar have grown to sufficient size that it is now possible to see that the colonies are uptaking a small amount of the red dye - which possibly suggests that the two separated parts of OBP2A are interacting. This will be shown through the use of the betagalactosidae assay. Cells of MG1655(-cya) from yesterdays overnight were firstly made competent then subsequently transformed with pUT18-OBP2A.
Protocols Used: Miniprep Pre-sequence digest p>
Results: N/A
Next Steps: If the transformation of MG1655(-cya) with pUT18-OBP2A is successful then the next step of experimentation will be retransform positive colonies with pT25-OBP2A.
Summary
Aim of Experiment: to test the hypotehesis that if a protein is seperated and the parts are reintrduced in vivo will they recombine, we have prepared a second attempt at performing the betagalactosidase assay in order to test the interaction of the two protein parts.
Protocols Used: Betagalctosidase assay
Next Steps: The next stage of experimentaton 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 betagalctosiase assay will be performed however this time it will be done under anerobic conditions.
Aim of Experiment: Performed a PCR of OBP2A using primers for pU1T18 and pT25 insertion which have been designed to place the smaller OBP2A into pUT18 and the larger subunit into pT25.
Protocols Used: PCR p>
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 into pUT18 and pT25.
Aim of Experiment: The PCR performed yesterday 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 OBP2Ain pUT18 and pT25.
Aim of Experiment: Asecond attempt at preparing OBP2A for insertion into the two vectors of te 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 betagalcatosidae assay, that will be done under anaerobic conditions.
Protocols Used: N/A p>
Results: N/A
Next Steps: The next stage of experimentation is to prepare for a anerobic betagalctosidase assay of my OBP2A in pT25 and pUT18 in the orignally designed conformation of large subunit in pUT18 and the smalled subunit in pT25.
Summary
Aim of Experiment: Prepared the samples of OBP2A for the anerobic betagalctosidase assay, so they can be frozen overnight in preperation for the assay itself that will be done tomorrow.
Protocols Used: Assay Preperation
Results:
Next Steps: The next stage of experimentation will be to perform the betagalctosidase assay itself, this will most likely be done tomorrow.
Aim of Experiment: Performed an anaerobic betagalctosidase 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: Betagalctosidae assay p>
Results: Must add in results.
Next Steps: As the results have shown that the two subunits of OBP2A in fact don't seem to be interacting in vivo - a possible reason might be aggregation of the two subunits preventing interaction. Then the next stage of experimentation will be to test out the interaction when the subunit are switch with regards to the vectors pUT18 and pT25. This will be the goal of the following weeks once the team is back from London.
