Team:Sevilla/Week 7

From 2011.igem.org

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<p>Great news, we must have left some food in the dustbin and the lab has been invaded by an army of ants! So instead of </p>
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<p>Great news, we must have left some food in the dustbin and the lab has been invaded by an army of ants! So it takes us some time to clean them all before starting to purify some digestions. We've introduced several changes in the protocol, following the advice of people from the department of Genetics. Let's hope it goes better, because we're starting to lose patiente...</p>
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<p>In the mean time, we try to transform some of our ligations...again. </p>
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
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<p>Tuesday 9 August</p>
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<p>Tuesday 23 August</p>
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The purifications from gel didn't go as expected, and to make matters worse the whole department of Genetics is in a complete chaos; there's no floor, all the important machines are hidden, and we don't have access to most of the rooms. It seems it won't get any better during the week...
+
We're giving up ethanol purification, it deffinitely doesn't work as it should. This means we have to re-define everything in order to start with the standard assembling, so that we only have to purify directly from the gel. Designing new digestions means having into account the size of every insert, where to cut them, in which order, which will be the vector, etc. so we spend the whole evening examining all the details carefully in order not to spoil it all tomorrow.
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
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<p>Wednesday 10 August</p>
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<p>Wednesday 24 August</p>
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A freshly cemented corridor separates us from the door behind which our PCR reactions are waiting for us to rescue them. Besides, getting to the NanoDrop or the Transiluminator rooms has become an adventure due to the piles of rubble and the hanging wires we have to dodge. This department has become a modern jungle, so we try not to come over here, and stay in our downstairs lab working on more digestiones and electrophoresis.
+
So, standard assembling, there we go. Lots of digestions with the corresponding inoculums to regenerate the mini-preps we've spent. Heat shock, electrophoresis,transiluminator...the same story...and yet we're supposed to have a good command of this protocols, the results are not always as expected. </p>
 +
<img src="https://lh6.googleusercontent.com/-RVrAbgnODTo/TnptLmYx3wI/AAAAAAAAA9w/1nJgQ0N-Y2A/s288/2011-08-19%25252011.23.08.jpg" style=padding-left:350px;" height="109" width="288" /></a>
 +
<p>And things are not going better with some easy stuff like transforming with a BioBrick...we're not able to get T9002, but where's the problem? Our competent cells? Our transformation protocol? The BioBrick from the kit? What the...?
 +
</p>
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<p>Science is not an exact science, no matter what people tell you...
</p>
</p>
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
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<p>Thursday 11 August</p>
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<p>Thursday 25 August</p>
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The department of Genetics is hardly accessible today, but things aren't better here in the lab: our fridge doesn't work properly and now we have all our chemicals and DNA samples surrounded by blocks of ice.  
+
We finally got to transform T9002, using different competent cells and a slightly different protocol. It will be useful for the project but firstly we need to test it. It's supposed to detect C6-3-oxo-HSL and generate GFP, but which concentration, how long it needs to be exposed to the signal...we have no clue about that. We'll have to design some basic experiments, but before that, we need to know whether our AHLs are OK, since they came a month late and probably not in the best conditions, given the 45ºC of Seville these days...</p>
-
</p>
+
<p>So we go upstairs, but not to disturb the people from de department of Genetics, but the microbiologists in the third floor this time. We've heard of a bacterium, Chromobacterium violaceum, that can detect a wide range of AHLs and turn violet. This will be the perfect indicator. Charo Espuny kindly seeds for us a plate with C. violaceum 026 and gives us some useful instructions to start testing it. But for now, let's leave it growing o/n...</p>
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<p> How to fix a freezer, lesson one: gather some mops, ice trays to keep things cool and something to crush the blocks until they come unstuck. The rest is easy: open the freezer's door, put your chemicals in the trays, and then charge, charge, charge at the blocks and let the ice melt. Then clean up all the mess and VOILÀ, you'll have plenty of space for all your digestions, biobricks, etc.
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<p>In. Mid vel vel elementum, placerat tincidunt, vut lacus purus nunc massa a habitasse? Ac pid elementum aliquam, natoque! Porttitor facilisis amet lacus, ridiculus nascetur! Sociis vel aenean natoque penatibus aliquam in, augue mauris, dictumst, nunc mid platea a! Massa? Vut dis purus. Odio magna nec. Lorem ut turpis adipiscing scelerisque dis, scelerisque. Sed quis, porttitor et pulvinar in. Massa sit, porta urna nunc auctor non elementum! Augue dolor cum sociis, dignissim elementum. Integer. Penatibus. Phasellus et porta nec! Hac in nec porttitor ridiculus rhoncus. Hac natoque mid est nunc platea nisi sed habitasse! Amet nec placerat ultricies egestas.
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
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<p>Friday 12 August</p>
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<p>Friday 26 August</p>
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We finally got some ligations right, but they're too few and we're not sure whether they're OK. We'll have to study them carefully...
+
As half of the group is studying for their exams of September, the girls take control of the lab and put some order - this was becoming quite a jungle with pictures of electrophoresis, empty racks, notebooks and gloves all over the place.
</p>
</p>
 +
<p>While we keep trying to get our digestions and ligations right - the endless, tedious cycle of mini-prep, digestion, electrophoresis, purification, ligation, transformation... - we also dedicate our time to C. violaceum and the AHLs. We've seeded the strain in plates and added drops of our AHLs with different concentrations. First we try C6-3-oxo-HSL and C4-HSL. C12-3-oxo-HSL needs to wait until we know whether C6 is alright.</p>
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<strong>C. violaceum 026 test:</strong>
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Vel cum diam, lorem integer et montes, nec placerat, turpis lacus nec placerat, rhoncus rhoncus, a porta augue ac! Et aliquet. Porttitor diam egestas, non vut etiam montes, aliquet? Pellentesque. Dolor nascetur amet. Adipiscing sed pulvinar cursus! Urna in, in turpis duis in sit placerat, vut non aliquam? Mattis habitasse augue mus. Pulvinar auctor natoque est lacus porta sagittis! A in massa lundium? In non, lundium nisi magna proin elementum sed porttitor sed amet risus a aliquet mid a? Proin turpis, ac magna, dictumst lorem tincidunt penatibus sociis rhoncus? Duis cursus sit diam amet nisi mattis egestas habitasse diam.
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<p>1.Prepare an inoculum with several colonies of C. violaceum and leave it growing at 29ºC for a couple of hours.</p>
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<p>2.Then take about 200 ul of that culture and extend them all over the plate until it's dry.</p>
 +
<p>3.Add three drops of 5, 15 and 25 ul of a solution of C6-3-oxo-HSL 5 ug/ml in absolute methanol.</p>
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<p>4.Leave growing o/n at 29ºC.</p>
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Latest revision as of 01:39, 22 September 2011


Monday 22 August


Great news, we must have left some food in the dustbin and the lab has been invaded by an army of ants! So it takes us some time to clean them all before starting to purify some digestions. We've introduced several changes in the protocol, following the advice of people from the department of Genetics. Let's hope it goes better, because we're starting to lose patiente...

In the mean time, we try to transform some of our ligations...again.


Monday Protocols



Tuesday 23 August


We're giving up ethanol purification, it deffinitely doesn't work as it should. This means we have to re-define everything in order to start with the standard assembling, so that we only have to purify directly from the gel. Designing new digestions means having into account the size of every insert, where to cut them, in which order, which will be the vector, etc. so we spend the whole evening examining all the details carefully in order not to spoil it all tomorrow.


Tuesday Protocols



Wednesday 24 August


So, standard assembling, there we go. Lots of digestions with the corresponding inoculums to regenerate the mini-preps we've spent. Heat shock, electrophoresis,transiluminator...the same story...and yet we're supposed to have a good command of this protocols, the results are not always as expected.

And things are not going better with some easy stuff like transforming with a BioBrick...we're not able to get T9002, but where's the problem? Our competent cells? Our transformation protocol? The BioBrick from the kit? What the...?

Science is not an exact science, no matter what people tell you...


Wednesday Protocols



Thursday 25 August


We finally got to transform T9002, using different competent cells and a slightly different protocol. It will be useful for the project but firstly we need to test it. It's supposed to detect C6-3-oxo-HSL and generate GFP, but which concentration, how long it needs to be exposed to the signal...we have no clue about that. We'll have to design some basic experiments, but before that, we need to know whether our AHLs are OK, since they came a month late and probably not in the best conditions, given the 45ºC of Seville these days...

So we go upstairs, but not to disturb the people from de department of Genetics, but the microbiologists in the third floor this time. We've heard of a bacterium, Chromobacterium violaceum, that can detect a wide range of AHLs and turn violet. This will be the perfect indicator. Charo Espuny kindly seeds for us a plate with C. violaceum 026 and gives us some useful instructions to start testing it. But for now, let's leave it growing o/n...


Thursday Protocols



Friday 26 August


As half of the group is studying for their exams of September, the girls take control of the lab and put some order - this was becoming quite a jungle with pictures of electrophoresis, empty racks, notebooks and gloves all over the place.

While we keep trying to get our digestions and ligations right - the endless, tedious cycle of mini-prep, digestion, electrophoresis, purification, ligation, transformation... - we also dedicate our time to C. violaceum and the AHLs. We've seeded the strain in plates and added drops of our AHLs with different concentrations. First we try C6-3-oxo-HSL and C4-HSL. C12-3-oxo-HSL needs to wait until we know whether C6 is alright.


Friday Protocols


C. violaceum 026 test:

1.Prepare an inoculum with several colonies of C. violaceum and leave it growing at 29ºC for a couple of hours.

2.Then take about 200 ul of that culture and extend them all over the plate until it's dry.

3.Add three drops of 5, 15 and 25 ul of a solution of C6-3-oxo-HSL 5 ug/ml in absolute methanol.

4.Leave growing o/n at 29ºC.

Retrieved from "http://2011.igem.org/Team:Sevilla/Week_7"