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Team:Valencia/notebook
From 2012.igem.org
Notebook
First day
29-6-2012
Finally the Project has finally and oficially started with a meeting before the lab work. It seems that after so many reunions and meetings on the weekends, seems we have the ultimate work distribution and despite, many of us won’t be able to be at full time due to retake exams, and it seems the real beginning will have to wait at least one more week, it finally seems that all our crazy ideas will take form. Still are many issues to mend: for example our human practises’ blog still has not began, and the materials order we listed like a month ago still has not been ordered by our advisor. Anyway we hope everything keeps on rolling okay from now on. At least the Synechococcus strain we thought we had lost has been already found.
Week 1
9-7-2012
Our first lab day could be the dictionary definition of disaster. Why are we saying this? Oh, just for a few things: the materials order still has not been done and we’ve been announced that in case we use and finish any reactant or material from the university stock, we will have to pay for it (hooray!). The advisor has been at the lab supervising for only a couple hours, and he was gone when we went to the autoclave in order to sterilize some reactants to make BG-11 medium … Surprise!- there was bacteria growing all inside the autoclave… Goddamm … that was bloody stinking… and the lab technician didn’t know anything about it.
10-7-2012
We expected that now the lab technician was aware our problem with the autoclave will somehow help us to solve it… but as Murphy’s law states, if anything has got any chance of going wrong, it will sure go wrong and in case it’s already wrong, it will become even worse. The bacterial growth still was there when we came, but the lab technician wasn’t. Later we were told that the university was inaugurating a new research centre in Calpe (still inactive), and he was needed there. Later on we asked a university teacher for some permission to use the autoclave. Guess what? It was denied. Ah, and one more thing, due to the inauguration, in order to make the new centre look productive, our spectrophotometer has been brought there. We still don’t know when it’s going to be brought back to where it once belonged. Anyway we’ve managed to clean the autoclave by ourselves. It’s something.
11-7-2012
Today we’ve finally sterilized the reactants for the BG-11 medium for Synechococcus, except the manganese which is essential for making the bacteria grow, so we will have to replace it with a commercial fertilizer until the new materials order we made arrives –the same one which has not been ordered yet-. Meanwhile we’ve received new rumors: like it happened the year before, it seems that the university –and in consequence our lab- will be opened during August only till 2 p.m. (yoohoo!! Who needs more time?!) and the advisor who’s been in charge of opening our lab and making some supervision will take his holiday on next Friday and no replacement is known (UPDATE: finally it seems we have the permission to access to the lab… 1 out only-God-knows problems solved).
12-7-2012
Today we’ve made a first attempt to make our Synechococcus wild type strain grow in the lab. We still don’t know which way will be the most efficient and successful for our purposes, so we will try two ways (both of them using both our BG-11 medium broth supplemented with the fertilizer and without this supplementation). On the first attempt we will grow the coccus will be grown in a flask sealed with parafilm which will receive all the aeration it needs with an air pump from a home aquarium; and on our second attempt, we will close the flask with hydrophobic cotton and we will grow it in an agitation chamber. Both attempts will be done with constant light with a lamp placed ant a 30 cm distance. We will have to wait until the culture has grown to determine which way is the most efficient. Weird news of the day: there is no more bad news.
13-7-2012
Our engineers think they’ve found a way to improve the growing of the Synechococcus culture. They have decided to make a CO2 bomb with a culture with yeast and sugar connected to the main culture so all the carbon dioxide produced by the yeast arrives to the cyanobacteria culture to boost the growing. Everything we have needed for this was a block of baking yeast, a couple of plastic tubes sterilized with 70º ethanol, some sugar and a soda bottle, it may not look very professional but if it works, who cares? Just hope it works when it’s done.
14-7-2012
As we expected, no bacterial growth has been observed at the flasks we’ve put to grow (according to bibliography, Synechococcus grows extremely slowly in comparison to other microorganisms like E.coli reaching its highest growing in which is in its optimal to transform in approximately one week). Nothing wrong has happened today? Well it almost happened (or at least we hope so): the temperature of the agitation chamber got decontrolled and reached almost 30 degrees (the optimal temperature of growing for our strain is approximately of 25 degrees) and it has been that high for some hours.
Week 2
16-7-2012
At this point, we’re supposed to observe the Synechococcus growing at the flasks, and as we expected after the series of unfortunate events that took place the last week no bacterial growing was seen at any flask at all, just as expected. It seems that the heat excess killed all the Synechococcus, so we will have to begin again. At least now we’ve got a list of what must not be done.
17-7-2012
Today’s been a real quiet day, we’ve prepared new BG-11 medium broth in order to regrow the bacteria (also we are not sure that pH of the last days medium is the best for the bacteria). After weighting the salts for the media, we’ve been told that the scales we’ve been using are not calibrated properly. Anyway we put those salts diluted in 100 ml at the autoclave.
18-7-2012
Finally, after making some considerations, the CO2 pump that our engineers designed the last week is finally ready to be used. So we will try in parallel the two growing conditions: at the agitation chamber with the hydrophobic cotton sealing the flask or the air bomb and the CO2 pump. For ensuring that the experiment is successful we also have prepared new BG-11 medium (the fact that the medium that we made the days before was at a 3,35 pH while the proper one is 7.5 has nothing to do with it, anyway apart from suspect that the pH-meter needs also to be calibrated, we have prepared a NaOH 2 M solution in order to highen it).
19-7-2012
Finally it seems that our BG-11 medium is properly done, we can observe some green colour at the flasks we put yesterday, but as is becoming usual bad news incoming. There’s an spectrophotometer aviable on the third floor of the university but as we expected, we’re banned (they plead that it’s from a private company so the university can’t say anything about it usage) so we will have to use a Neubauer chamber in order to count the culture density. Ah… almost forgot, we’ve been reprehended because of using the balance (the same ones which were discalibrated… any responsible?).
20-7-2012
The culture seems to be working as expected, the counting on the Neubauer chambers give the following results despite of the little time the culture has been there. Specifically we’ve observed an amount of 40 cfu/L in the medium nº 1 (BG 11 , pH=7,36 without micronutrients and with the CO2 pump) and 26 cfu/L in the medium nº2 (BG 11, pH= 8,36 with micronutrients)
Week 3
23-7-2012
Again we count the cfu/l from 3 different mediums, having these three different results in medium nº1 (BG-11, pH 7.36, without micronutrients ) we had 350,5 CFU/L; in medium nº 2 (BG-11, pH 8.18 with micronutrients) we had 343,25 CFU/L while in medium nº 3 (BG-11 + fertilizer, pH 7.3 with the C0 2 pump)= 2420 CFU/L. We’ve prepared and sterilized more BG-11 in order to subcultivate when it arrives to its maximum.
24-7-2012
The counting today seems to give the following results: 695 CFU/L for the medium nº1, 875 CFU/L for the medium nº2 and 8381 CFU/for medium nº 3 (which also presents a strange colour, mixing white and green). Due to these limits, we’ve decided to make only qualitative measures despite of quantitative.
25-7-2012
It seems that the flasks with mediums 1 and 2 have began to enter into their final phase, while no changes are observed on flask with medium nº 3.
26-7-2012
No coccus remains on the medium nº 1 while it still seems we have some on medium nº2.
27-7-2012
The expedition to Alicante has been successful. We’ve managed to get a plasmid with the LuxAB gene (but without the promoter) and also have given us some piece of advice about how to grow Synechococcus: basically grow it in sterile conditions, only needs to be in a culture chamber with mechanic agitation.
Week 4
Description
Week 5
Description
Week 6
Description
Week 7
Description
Week 8
Description
Week 9
Description
Week 10
Description
Week 11
Description
Week 12
Description
9-7-2012
Our first lab day could be the dictionary definition of disaster. Why are we saying this? Oh, just for a few things: the materials order still has not been done and we’ve been announced that in case we use and finish any reactant or material from the university stock, we will have to pay for it (hooray!). The advisor has been at the lab supervising for only a couple hours, and he was gone when we went to the autoclave in order to sterilize some reactants to make BG-11 medium … Surprise!- there was bacteria growing all inside the autoclave… Goddamm … that was bloody stinking… and the lab technician didn’t know anything about it.
10-7-2012
We expected that now the lab technician was aware our problem with the autoclave will somehow help us to solve it… but as Murphy’s law states, if anything has got any chance of going wrong, it will sure go wrong and in case it’s already wrong, it will become even worse. The bacterial growth still was there when we came, but the lab technician wasn’t. Later we were told that the university was inaugurating a new research centre in Calpe (still inactive), and he was needed there. Later on we asked a university teacher for some permission to use the autoclave. Guess what? It was denied. Ah, and one more thing, due to the inauguration, in order to make the new centre look productive, our spectrophotometer has been brought there. We still don’t know when it’s going to be brought back to where it once belonged. Anyway we’ve managed to clean the autoclave by ourselves. It’s something.
11-7-2012
Today we’ve finally sterilized the reactants for the BG-11 medium for Synechococcus, except the manganese which is essential for making the bacteria grow, so we will have to replace it with a commercial fertilizer until the new materials order we made arrives –the same one which has not been ordered yet-. Meanwhile we’ve received new rumors: like it happened the year before, it seems that the university –and in consequence our lab- will be opened during August only till 2 p.m. (yoohoo!! Who needs more time?!) and the advisor who’s been in charge of opening our lab and making some supervision will take his holiday on next Friday and no replacement is known (UPDATE: finally it seems we have the permission to access to the lab… 1 out only-God-knows problems solved).
12-7-2012
Today we’ve made a first attempt to make our Synechococcus wild type strain grow in the lab. We still don’t know which way will be the most efficient and successful for our purposes, so we will try two ways (both of them using both our BG-11 medium broth supplemented with the fertilizer and without this supplementation). On the first attempt we will grow the coccus will be grown in a flask sealed with parafilm which will receive all the aeration it needs with an air pump from a home aquarium; and on our second attempt, we will close the flask with hydrophobic cotton and we will grow it in an agitation chamber. Both attempts will be done with constant light with a lamp placed ant a 30 cm distance. We will have to wait until the culture has grown to determine which way is the most efficient. Weird news of the day: there is no more bad news.
13-7-2012
Our engineers think they’ve found a way to improve the growing of the Synechococcus culture. They have decided to make a CO2 bomb with a culture with yeast and sugar connected to the main culture so all the carbon dioxide produced by the yeast arrives to the cyanobacteria culture to boost the growing. Everything we have needed for this was a block of baking yeast, a couple of plastic tubes sterilized with 70º ethanol, some sugar and a soda bottle, it may not look very professional but if it works, who cares? Just hope it works when it’s done.
14-7-2012
As we expected, no bacterial growth has been observed at the flasks we’ve put to grow (according to bibliography, Synechococcus grows extremely slowly in comparison to other microorganisms like E.coli reaching its highest growing in which is in its optimal to transform in approximately one week). Nothing wrong has happened today? Well it almost happened (or at least we hope so): the temperature of the agitation chamber got decontrolled and reached almost 30 degrees (the optimal temperature of growing for our strain is approximately of 25 degrees) and it has been that high for some hours.
Week 2
16-7-2012
At this point, we’re supposed to observe the Synechococcus growing at the flasks, and as we expected after the series of unfortunate events that took place the last week no bacterial growing was seen at any flask at all, just as expected. It seems that the heat excess killed all the Synechococcus, so we will have to begin again. At least now we’ve got a list of what must not be done.
17-7-2012
Today’s been a real quiet day, we’ve prepared new BG-11 medium broth in order to regrow the bacteria (also we are not sure that pH of the last days medium is the best for the bacteria). After weighting the salts for the media, we’ve been told that the scales we’ve been using are not calibrated properly. Anyway we put those salts diluted in 100 ml at the autoclave.
18-7-2012
Finally, after making some considerations, the CO2 pump that our engineers designed the last week is finally ready to be used. So we will try in parallel the two growing conditions: at the agitation chamber with the hydrophobic cotton sealing the flask or the air bomb and the CO2 pump. For ensuring that the experiment is successful we also have prepared new BG-11 medium (the fact that the medium that we made the days before was at a 3,35 pH while the proper one is 7.5 has nothing to do with it, anyway apart from suspect that the pH-meter needs also to be calibrated, we have prepared a NaOH 2 M solution in order to highen it).
19-7-2012
Finally it seems that our BG-11 medium is properly done, we can observe some green colour at the flasks we put yesterday, but as is becoming usual bad news incoming. There’s an spectrophotometer aviable on the third floor of the university but as we expected, we’re banned (they plead that it’s from a private company so the university can’t say anything about it usage) so we will have to use a Neubauer chamber in order to count the culture density. Ah… almost forgot, we’ve been reprehended because of using the balance (the same ones which were discalibrated… any responsible?).
20-7-2012
The culture seems to be working as expected, the counting on the Neubauer chambers give the following results despite of the little time the culture has been there. Specifically we’ve observed an amount of 40 cfu/L in the medium nº 1 (BG 11 , pH=7,36 without micronutrients and with the CO2 pump) and 26 cfu/L in the medium nº2 (BG 11, pH= 8,36 with micronutrients)
Week 3
23-7-2012
Again we count the cfu/l from 3 different mediums, having these three different results in medium nº1 (BG-11, pH 7.36, without micronutrients ) we had 350,5 CFU/L; in medium nº 2 (BG-11, pH 8.18 with micronutrients) we had 343,25 CFU/L while in medium nº 3 (BG-11 + fertilizer, pH 7.3 with the C0 2 pump)= 2420 CFU/L. We’ve prepared and sterilized more BG-11 in order to subcultivate when it arrives to its maximum.
24-7-2012
The counting today seems to give the following results: 695 CFU/L for the medium nº1, 875 CFU/L for the medium nº2 and 8381 CFU/for medium nº 3 (which also presents a strange colour, mixing white and green). Due to these limits, we’ve decided to make only qualitative measures despite of quantitative.
25-7-2012
It seems that the flasks with mediums 1 and 2 have began to enter into their final phase, while no changes are observed on flask with medium nº 3.
26-7-2012
No coccus remains on the medium nº 1 while it still seems we have some on medium nº2.
27-7-2012
The expedition to Alicante has been successful. We’ve managed to get a plasmid with the LuxAB gene (but without the promoter) and also have given us some piece of advice about how to grow Synechococcus: basically grow it in sterile conditions, only needs to be in a culture chamber with mechanic agitation.
Week 4
Description
Week 5
Description
Week 6
Description
Week 7
Description
Week 8
Description
Week 9
Description
Week 10
Description
Week 11
Description
Week 12
Description
