Where the first week was mainly introduction to the iGEM project where we had many meetings, this week was full of ideas, lab work and research. We split into teams to do lab work and research and came together at the end and start of the day to exchange information and ideas. The week was very productive in all aspects. We finally got the transformations to work after many attempts. We realise that throughout the coming weeks, there will be many challenges which we gladly will take on solve.
+
Where the first week was mainly introduction to the iGEM project, many meetings and planning, this week was full of ideas, lab work and research. We split into teams dedicated to lab work and research and came together at the end and start of each day to exchange information and ideas. The week was very productive in all aspects. After many attempts we finally got the transformations to work. We realise that throughout the coming weeks, there will be many challenges which we gladly will take on.
-
==Day 1==
+
==Day 1 (16/07/12)==
===Research===
===Research===
-
While two members of the team worked in the labs on the transformation of ''Escherichia coli'' the rest of the team continued with the research into previous BioBricks and those that we felt the characterisation of would be useful to our project. As one of our proposed ideas for the project is to produce a BioBrick that can sense nitric oxide and then go on to increase nitric oxide amounts to toxic levels if appropriate as a potential cancer therapeutic we decided to look into genes for nitric oxide synthase and nitrite reductase, both enzymes that result in the production of nitric oxide. We also looked into plasmids and genes that may be used in mammalian cells as well as bacterial in order to start thinking about where our project could go once the BioBrick is synthesised.
+
While two members of the team worked in labs on the transformation of ''Escherichia coli'', the rest of the team continued with research into previous BioBricks that we felt characterising would be useful to our project. One of our proposed ideas for the project was to design a BioBrick that can sense nitric oxide and go on to increase nitric oxide to toxic levels utilising nitric oxide synthases (NOSs) to develop a potential cancer therapeutic. We decided to look further into NOSs and nitrite reductases, both enzyme families that result in the production of nitric oxide. We also looked into plasmids and genes that may be used in mammalian cells as well as bacterial, in order to start thinking about where our project could go, once the BioBricks were synthesised. Since we were working with a hybrid promoter of bacterial and mammalian cells and the idea to use a salmonella chassis, presented to us by Sebastian Runkel last week, we hypothesised that this route might pose as a very interesting area to develop.
===Labs===
===Labs===
-
Transformation of the three BioBricks was redone. Two controls were also set up in order to point us in the right direction with what may have gone wrong; the positive control used BRAF (BL21 pLysS) cells which contain vectors for chloramphenicol resistance; the negative control used the NEB 5-alpha cells used in the previous experiment; our aim was for the positive control to show whether the issue lies within the chloramphenicol resistance, or whether the problem lies within the competent cells themselves.
+
We started with repeating the transformation of the three Biobricks used last week into NEB 5-alpha ''Escherichia coli'', utilising adjusted protocols sprung from our aim to improve the success rate. Two controls were set up in order to point us in the right direction with what may have gone wrong; the positive control used was BRAF (BL21 pLysS) cells which contain vectors for chloramphenicol resistance; the negative control was NEB 5-alpha cells on their own. Our aim was for the positive control to show whether the issue lied within the chloramphenicol resistance, or whether the problem lied within the competent cells themselves.
-
A sample of lysogeny broth (LB) was also inoculated with premade culture containing the BL21 pLysS cells in preparation for the next day's plasmid isolation focusing on chloraphenicol resistance.
+
A sample of lysogeny broth (LB) was also inoculated with a premade culture containing BL21 pLysS cells in preparation for the next day's plasmid isolation, focusing on obtaining plasmids giving chloramphenicol resistance.
-
Protocols for the experiments can be found [[Team:NRP-UEA-Norwich/Protocol | Here]]
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Protocols for the experiments can be found [[Team:NRP-UEA-Norwich/Protocol | here.]]
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==Day 2 (17/07/12)==
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==Day 2==
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===Research===
===Research===
-
As we are looking at producing a potential cancer therapeutic as part of our project we focused on looking at how that particular application could be used. Our main idea is to attach a nitric oxide-sensing promoter to an enzyme producing more nitric oxide (NO) in order to bring NO levels to a toxic threshold. We have found two key classes of NO-producing enzyme that may be applicable to our project: the first is nitric oxide synthase (NOS), most commonly from ''Nocardia'' species (known as NOSNoc) as it is a bacterial form of NOS, however so far we found little information on the intricacies of the enzyme and therefore feel it may not be the best enzyme to use; the other enzyme we have looked at is nitrite reductase (NiR), specifically copper-based NiR as it has a much cleaner reaction mechanism (producing just NO and H+) compared with other species of NiR.
+
As we were looking at producing a potential cancer therapeutic as part of our project, we focused on researching how that particular application could be used in the future. Our main goal was to attach a nitric oxide-sensing promoter to an enzyme that will increase the nitric oxide (NO) concentration in order for it to reach a toxic threshold. We had found two key classes of NO-producing enzyme, that may be applicable to our project: the first was nitric oxide synthases (NOSs), most commonly from ''Nocardia'' species (known as NOSNoc) as it is a bacterial form of NOS. However so far we had found little information on the intricacies of the enzyme and therefore felt it may not be the best enzyme to use; the other enzyme we had considered was nitrite reductase (NiR), specifically copper-based NiR, as it has a much cleaner reaction mechanism (producing just NO and H+) compared with other species of NiR.
===Labs===
===Labs===
-
Rebecca and Lukas went back into the labs today in order to try and transform the ''E. coli'' once again. Using the knowledge we had gained through research into why the previous transformations had been unsuccessful agar was used that had a reduced amount of chloramphenicol (50µg/ml to 25µg/ml) in order to try and grow more cells.
+
Rebecca and Lukas went back into labs today in order to try and transform the ''E. coli'' once again. Using the knowledge we had gained through research into why the previous transformations had been unsuccessful, agar with a reduced concentration of chloramphenicol (50µg/ml to 25µg/ml) was prepared. With minor adjustments to the protocol we hoped that this time the transformation was successful.
-
We were also visited by Josh Wright from Bioline who very kindly donated a large amount of plasmid isolation kits and PCR gel clean-up kits, as well as giving us a discount on Bioline products. This kind of support from Bioline is extremely important to our project and we are very thankful!
+
We were also payed a visit by Josh Wright from Bioline, who very kindly donated a large amount of plasmid isolation and PCR gel clean-up kits, as well as giving us a discount on Bioline products. This kind support from Bioline was extremely important to our project and we were very thankful!
-
Following Josh's visit Lukas, Russell and Khadija decided to try out the plasmid isolation kits for the first time as they tried to extract the chloramphenicol-resistance plasmid from some cultured ''E. coli''. The kits were extremely easy to use, however in keeping with our previous problems with growing ''E. coli'' we were unable to extract the plasmid, which was proven to us by a blank agarose gel picture.
+
Following Josh's visit, Lukas, Russell and Khadija decided to try out the plasmid isolation kits for the first time with the extraction of a chloramphenicol-resistance plasmid from the BL21 pLysS cell culture inoculated yesterday. The kits were easy to use, however in keeping with our previous problems with growing ''E. coli'', we were unable to extract any plasmid DNA, which was proven to us by a blank agarose gel picture. With research into the origin of the fault, we troubleshot the protocol and were hopeful to perform a successful mini prep tomorrow.
-
The protocol for all lab work can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Here]
+
The adjusted protocols for all lab work can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol here.]
-
==Day 3==
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==Day 3 (18/07/12)==
===Labs===
===Labs===
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Following the blank agarose gel image more E.coli was inoculated into culture in hope that the lack of DNA was due to an insufficiency in the number of cells.
-
A/B: Cells with Bba_K381001 + pSB1C3 plasmid (20µl and 200µl)
+
Following the blank agarose gel image of the plasmid DNA isolation, more BL21 pLysS cells were inoculated into culture in hope that the lack of DNA was due to an insufficiency in the cell number.
-
C: Cells with Bba_K325100 + pSB1C3 plasmid (200µl)
+
We were very glad that the transformation from yesterday was successful and our positive control as well as the samples of the cells transformed with one of four BioBricks each, showed colonies of viable cells, that successfully grew. Following our experimental design, the negative control showed no cell growth at all.
+
Using the plates from this experiment, we inoculated LB cultures with cells from the following plates and they were incubated in preparation for mini preps tomorrow:
-
D: Cells with Bba_K325909 + pSB1C3 plasmid (small colonies)
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A/B: Cells with Bba_K381001 in pSB1C3 plasmid (20µl and 200µl)
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E/F/G: Cells with Bba_K381001 + pSB1C3 plasmid(200µl)
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C: Cells with Bba_K325100 in pSB1C3 plasmid (200µl)
+
+
D: Cells with Bba_K325909 in pSB1C3 plasmid (small colonies)
+
+
E/F/G: Cells with Bba_K381001 in pSB1C3 plasmid(200µl)
Positive control: colony of Cells with plys plasmid (Chloramphenicol resistant)
Positive control: colony of Cells with plys plasmid (Chloramphenicol resistant)
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==Day 4==
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==Day 4 (19/07/12)==
===Labs===
===Labs===
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[[File:First_mini_prep right orientation.jpg | thumb | '' '''Figure 1.''' Gel electrophoresis of one of our first Mini preps.'' ]]
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We looked at the cultures grown overnight and found some grew better than others. The cells growing in tubes A,B and the four controls showed good visible growth. However, the other tubes showed no growth and thus were incubated for longer. Later, it was found that tube G showed bacterial growth but the other tubes didn't.
+
We observed the cultures grown overnight and found that some grew better than others. The cells growing in tubes A, B and the four controls showed good visible growth. However the other tubes showed no growth and thus were incubated for longer. Later, it was found that tube G showed bacterial growth but the other tubes didn't.
-
Joy and Rachel isolated the DNA, changing the volumes used to plasmid high copy number volumes. We were elated to find that after electrophoresis, there was isolated DNA. The wells containing DNA extracted from cells with K381001 + pSB1C3 plasmids show a similar three band pattern but of different intensity. This may be a result of varying concentrations of DNA. The
+
Joy and Rachel isolated the plasmid DNA from tubes A/B, adjusting the protocol to utilise volumes for high copy number plasmids to increase the concentration of the plasmid DNA in the final sample. We were elated to find that after electrophoresis, there was isolated DNA (''Figure 1.'').
-
DNA samples extracted from cells with plys plasmid show an identical pattern.
+
The samples containing DNA extracted from cells with K381001 + pSB1C3 plasmids, showed a similar three band pattern but of different intensity. This may have been a result of varying concentrations of DNA. The DNA samples extracted from cells with plys plasmid showed an identical pattern.
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and 6.
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The protocol can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Isolating_DNA_.28Bioline.29 here,]
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==Day 5==
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==Day 5 (20/07/12)==
===Labs===
===Labs===
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The hybrid promoters arrived and immediately we got to work on them. There were two promoters. We ordered two hybrid promoters composed of a mammalian and bacterial promoter which both reacts to NO. The difference in our two promoters was the orientation of the bacterial and mammalian components. We labelled them M-B (mammalian + bacterial-E9-ns2 - CArG + BHN + BBaK216005) and B-M (bacterial + mammalian - BBaK216005 + BHN + CArG - E9-ns2). They came in the dry form, which we hydrated and transformed.
+
The order of the synthesised hybrid promoters finally arrived and immediately we got to work. The synthesised genes consisted of two hybrid promoters made up of BBaK216005 and CArG - E9-ns2 in opposite orientation. Therefor we will be working with two genes composed of a mammalian and bacterial part which both react to NO. The only difference is the orientation of the components. To distinguish between them we will refer to them as M-B (mammalian + bacterial, E9-ns2 - CArG + BHN + BBaK216005) and B-M (bacterial + mammalian, BBaK216005 + BHN + CArG - E9-ns2). The DNA was shipped in dry form, which was hydrated and transformed using the optimised protocol developd over the last two weeks.
+
+
[[File:PyeaR+GFP_E+P.png | thumb | '' '''Figure 2.''' Restriction map of Pyear+GFP in pSB1C3 double digested with EcoRI and PstI.'']]
+
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The plasmid DNA, containing the PyeaR Bio-Brick, isolated 2 days ago was double digested using EcoR1 and Pst1 as to isolate the BioBrick PyeaR DNA sequence from the plasmid, beginning with the iGEM suffix and ending with the iGEM prefix (''Figure 2.'').
+
The restricted DNA was stored in the freezer for later confirmation of success via gel electrophoresis.
-
The plasmid containing the PyeaR Bio-Brick isolated a few days ago was cut using restriction enzymes. We used EcoR1 and Pst1 as these are in the suffix and prefix of the plasmid. The restricted DNA was stored in the freezer for later confirmation of success via gel electrophoresis.
+
Details of the protocol can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Single_Digest_for_validation here.]
Where the first week was mainly introduction to the iGEM project, many meetings and planning, this week was full of ideas, lab work and research. We split into teams dedicated to lab work and research and came together at the end and start of each day to exchange information and ideas. The week was very productive in all aspects. After many attempts we finally got the transformations to work. We realise that throughout the coming weeks, there will be many challenges which we gladly will take on.
Day 1 (16/07/12)
Research
While two members of the team worked in labs on the transformation of Escherichia coli, the rest of the team continued with research into previous BioBricks that we felt characterising would be useful to our project. One of our proposed ideas for the project was to design a BioBrick that can sense nitric oxide and go on to increase nitric oxide to toxic levels utilising nitric oxide synthases (NOSs) to develop a potential cancer therapeutic. We decided to look further into NOSs and nitrite reductases, both enzyme families that result in the production of nitric oxide. We also looked into plasmids and genes that may be used in mammalian cells as well as bacterial, in order to start thinking about where our project could go, once the BioBricks were synthesised. Since we were working with a hybrid promoter of bacterial and mammalian cells and the idea to use a salmonella chassis, presented to us by Sebastian Runkel last week, we hypothesised that this route might pose as a very interesting area to develop.
Labs
We started with repeating the transformation of the three Biobricks used last week into NEB 5-alpha Escherichia coli, utilising adjusted protocols sprung from our aim to improve the success rate. Two controls were set up in order to point us in the right direction with what may have gone wrong; the positive control used was BRAF (BL21 pLysS) cells which contain vectors for chloramphenicol resistance; the negative control was NEB 5-alpha cells on their own. Our aim was for the positive control to show whether the issue lied within the chloramphenicol resistance, or whether the problem lied within the competent cells themselves.
A sample of lysogeny broth (LB) was also inoculated with a premade culture containing BL21 pLysS cells in preparation for the next day's plasmid isolation, focusing on obtaining plasmids giving chloramphenicol resistance.
As we were looking at producing a potential cancer therapeutic as part of our project, we focused on researching how that particular application could be used in the future. Our main goal was to attach a nitric oxide-sensing promoter to an enzyme that will increase the nitric oxide (NO) concentration in order for it to reach a toxic threshold. We had found two key classes of NO-producing enzyme, that may be applicable to our project: the first was nitric oxide synthases (NOSs), most commonly from Nocardia species (known as NOSNoc) as it is a bacterial form of NOS. However so far we had found little information on the intricacies of the enzyme and therefore felt it may not be the best enzyme to use; the other enzyme we had considered was nitrite reductase (NiR), specifically copper-based NiR, as it has a much cleaner reaction mechanism (producing just NO and H+) compared with other species of NiR.
Labs
Rebecca and Lukas went back into labs today in order to try and transform the E. coli once again. Using the knowledge we had gained through research into why the previous transformations had been unsuccessful, agar with a reduced concentration of chloramphenicol (50µg/ml to 25µg/ml) was prepared. With minor adjustments to the protocol we hoped that this time the transformation was successful.
We were also payed a visit by Josh Wright from Bioline, who very kindly donated a large amount of plasmid isolation and PCR gel clean-up kits, as well as giving us a discount on Bioline products. This kind support from Bioline was extremely important to our project and we were very thankful!
Following Josh's visit, Lukas, Russell and Khadija decided to try out the plasmid isolation kits for the first time with the extraction of a chloramphenicol-resistance plasmid from the BL21 pLysS cell culture inoculated yesterday. The kits were easy to use, however in keeping with our previous problems with growing E. coli, we were unable to extract any plasmid DNA, which was proven to us by a blank agarose gel picture. With research into the origin of the fault, we troubleshot the protocol and were hopeful to perform a successful mini prep tomorrow.
The adjusted protocols for all lab work can be found here.
Day 3 (18/07/12)
Labs
Following the blank agarose gel image of the plasmid DNA isolation, more BL21 pLysS cells were inoculated into culture in hope that the lack of DNA was due to an insufficiency in the cell number.
We were very glad that the transformation from yesterday was successful and our positive control as well as the samples of the cells transformed with one of four BioBricks each, showed colonies of viable cells, that successfully grew. Following our experimental design, the negative control showed no cell growth at all.
Using the plates from this experiment, we inoculated LB cultures with cells from the following plates and they were incubated in preparation for mini preps tomorrow:
A/B: Cells with Bba_K381001 in pSB1C3 plasmid (20µl and 200µl)
C: Cells with Bba_K325100 in pSB1C3 plasmid (200µl)
D: Cells with Bba_K325909 in pSB1C3 plasmid (small colonies)
E/F/G: Cells with Bba_K381001 in pSB1C3 plasmid(200µl)
Positive control: colony of Cells with plys plasmid (Chloramphenicol resistant)
Day 4 (19/07/12)
Labs
Figure 1. Gel electrophoresis of one of our first Mini preps.
We observed the cultures grown overnight and found that some grew better than others. The cells growing in tubes A, B and the four controls showed good visible growth. However the other tubes showed no growth and thus were incubated for longer. Later, it was found that tube G showed bacterial growth but the other tubes didn't.
Joy and Rachel isolated the plasmid DNA from tubes A/B, adjusting the protocol to utilise volumes for high copy number plasmids to increase the concentration of the plasmid DNA in the final sample. We were elated to find that after electrophoresis, there was isolated DNA (Figure 1.).
The samples containing DNA extracted from cells with K381001 + pSB1C3 plasmids, showed a similar three band pattern but of different intensity. This may have been a result of varying concentrations of DNA. The DNA samples extracted from cells with plys plasmid showed an identical pattern.
The order of the synthesised hybrid promoters finally arrived and immediately we got to work. The synthesised genes consisted of two hybrid promoters made up of BBaK216005 and CArG - E9-ns2 in opposite orientation. Therefor we will be working with two genes composed of a mammalian and bacterial part which both react to NO. The only difference is the orientation of the components. To distinguish between them we will refer to them as M-B (mammalian + bacterial, E9-ns2 - CArG + BHN + BBaK216005) and B-M (bacterial + mammalian, BBaK216005 + BHN + CArG - E9-ns2). The DNA was shipped in dry form, which was hydrated and transformed using the optimised protocol developd over the last two weeks.
Figure 2. Restriction map of Pyear+GFP in pSB1C3 double digested with EcoRI and PstI.
The plasmid DNA, containing the PyeaR Bio-Brick, isolated 2 days ago was double digested using EcoR1 and Pst1 as to isolate the BioBrick PyeaR DNA sequence from the plasmid, beginning with the iGEM suffix and ending with the iGEM prefix (Figure 2.).
The restricted DNA was stored in the freezer for later confirmation of success via gel electrophoresis.
"
