First day

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

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.

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.

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).

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.

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.

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

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.

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.

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).

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?).

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

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 C02 pump)= 2420 CFU/L. We’ve prepared and sterilized more BG-11 in order to subcultivate when it arrives to its maximum.

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 nº3 (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.

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 nº3.

No coccus remains on the medium nº 1 while it still seems we have some on medium nº2.

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

Surprise! What we were growing on the only flask that seemed to work (the one of the CO2 pump) is not only Synechococcus but Synechococcus contaminated with yeast! It seems that when they both grow together they produce some kind of synergy and the yeast enhances the coccus growing (apparently the cause of the contamination is a failure on the aeration, surely due to the lack of a filter on the CO2 pump).

Finally the materials order has arrived. No molecular weight marker (which equals no chance of making an electrophoresis) still lack some micronutrients (manganese particularly) the restriction enzymes have been ordered three times (each one of them corresponding to each of the commercial brands we listed as possible choices) and we even have things we haven’t ordered like a gigantic bag with 50,000 corning tubes. I like when everything goes allright!. By the way, let’s say hello to the only in the morning lab days!

Here it is!! The most anticipated event of the summer!!! Finally among us… the morning only lab times!!!! (Despite of this, it doesn’t mean that during the afternoons and the evenings the work is gonna stop, in fact, the meetings at somebody’s flat is going to become a constant due to the current state of our wiki). Aside from this in the lab all the stuff is pretty quiet due to the lack of some materials that will keep on arriving during this week (which includes the molecular weight marker). Aside from this, we’ve begun to attempt growing some Synechococcus now the antibiotics have arrived.

Nothing else has arrived today. Labwork still pretty paralyzed; for example today the only thing we’ve been able to do is to transform E.coli with a Chlamydomona plasmid (still don’t know which method will be better for the biolamp, Synechococcus or Chlamydomonas reihardii , but it seems that the reunions at the projectmates’ flats are giving some interesting results in all the issues which are not related to the labwork. Logo designed (and we’ve to admit that it’s pretty awesome), name decided (we have to admit that some of the team members didn’t like it at all but they weren’t at the meeting where it was decided).

Time to do some transformations! First of all we will do some transformations with E.coli and the Part Registry BioBrick which codes for the IPTG gene in order to make some tests with the AHL gene (a step that will take longer than expected because we lack the ligase we had ordered).

Week 5

Some more good news after all: We’ve got the ligase. Finally seems we’re on the optimal conditions to do our work properly (if we forget about the time limitations imposed by the university). Now we will be able to began with the constructs. As we stated before, we will fist begin with a test growing E.coli and transfoming it with the IPTG and the AHL that we will use for making Allivibrio fischeri shine. Due to the fast growing of the bacteria and the fact that our university only allows us to work on the lab at the mornings, it’s kind of probably that the bacteria will be in a phase in which it’s not able to work with so we will try to slow down its growing with cold just after transforming.

7-8-2012 No bacteria available at the growing tubes. We wiil have to do it again, but we will have to find a way to do all the steps in only one morning.

New stuff has arrived today. It’s a special Synechococcus strain one of our members ordered from Harvard called cscB which seems to be able to export huge amounts of sucrose (in fact the people who have supplied us with this strain say that it’s the most efficient sugar exporting strain that we can find). Seems it will ease our job(or at least we hope so).

Today’s work session has been more focused on the wiki’s technical issues rather than in the lab stuff. We have decided the project’s final name and some esthetical stuff. Anyway, there’s much more work to do than we thought on a very first moment.

Second attempt for growing E.coli. Now we will try to let it grow during the whole weekend in cold and see if we have found the key.

Week 6

Finally E. coli seems to have grown. Now is time make some subcultures and do the minipreps, being able to do today only the first of the ones we have mentioned before. Step by step the road gets walked!

14-8-2012 Second step of the ones stated the day before. Nothing seems to have gone wrong during the steps (which is truly suspicious). Aside from this, we begin to compare diverse bibliography sources in order to find out which is the most accurate sequence for the psbA I gene promoter in order to upload its sequence to the parts registry.

No work has been done today due to a religious festivity (it’s a bank holidays’ day; this inactivity has got nothing to do with the fact of working at a Catholic university).

Only three people have been in the lab this morning (and no advisors, as it seems to become usual). Today what we’ve done consisted basically on a miniprep for the plasmid with our construct with the IPTG and AHL genes and grow the CscB Synechococcus strain that Alex (our member in Barcelona) got from Harvard. No results were displayed on the electrophoresis gel.

Time to do another attempt for the IPTG+ AHL miniprep. Only the band was found for the AHL genes and for the Chlamydomonas plasmids; but in the AHL carriers we find two different bands. We suspect there’s contamination because who loaded the DNA on the electrophoresis gene did not change the pipette after the use (not confirmed, we also think it may be due an effect of the plasmid.

Week 7

Due to the lack of success of the last attempt here we go again: Let’s grow some Synechococcus!!!! Now we will try both in solid medium and in broth medium, having two different varieties in the solid medium: we will try with an ordinary BG-11 medium (still without manganese) with a 3% agar and a BG-11 supplemented with some plants fertilizer (and also its correspondent 3% agar). On the other hand the 3 flasks we culture with BG 11 broth medium were supplemented with plants fertilizer 1 micro liter per each milliliter).

21-8-2012 Today we proceeded to culture and inoculate the remaining flask with the CsCB Syenchococcus strain and to prepare some medium for some of our transformed E.coli in order to make another minipreps. On the other hand we managed to discover which are the Chlamydomonas optimal growing conditions.

A pretty productive day, we autoclaved some BG-11 medium and some fertilizer in order to grow some WT Synechococcus (all the concentrations are the same employed on the attempt done three days ago). Apart from this, we managed to digest the AHL biobrick part from its plasmid with EcoRI and PstI and digesting the Chlamydomonas gene in two different ways (with PstI and with XbaI and XhoI). The electrophoresis gel resulted to be tot well done, so we are forced to repeat it tomorrow.

Today we have begun to make another attempt of growing Synechococcus and Chlamydomona. We find out which are the proper temperatures for growing each coccus strain (35ºC for the CsCB strain and 30ºC for the WT strain).

Today’s work briefing: growing three Petri dishes with LB and ampiciline of E.coli transformed with the IPTG-induced plasmid we got from Alicante.

Week 8

And there we go again ready to make new medium! (Mental note. This time keep it in the fridge).

28-8-2012 Again preparing new medium and making minipreps. No new results have appreared. Despite of everything (specially the lack of experimental characterization and the fact that the specific sequence had some little variations among the different authors) we’ve managed to upload the psbA part to the Parts Registry.

Electrophoresis for the IPTG-induced plasmid miniprep. Good results after all!!!

pH for new BG-11 medium adjusted and ready to be used in the new CsCB Synechococcus strain cultures we have prepared (it’s a shame we hadn’t enough time to do the Petri dishes, God heavens this stupid time limitation ends this week! (we had to say it).

Last day of stupid imposed from above time limitation! Could we say the day has been productive? Well, not much at all.

Week 9

Finally back on the regular schedule (the one that should had been all this time). And FINALY some coccus seems to have grown (two Petri dishes from the CsCB strain the cultured the last Friday at a 8.9 pH and some on the flask we left on the stove). Now we’ve prepared some Petri dishes with LB medium in order to culture E.coli transformed with a plasmid we obtained from Susan Golden; diluted some plants fertilizer to make more coccus subcultures and prepare some TE buffer. Later in the evening, we’ve transformed with the Susan Golden plasimid in order to make a test, make the electrophoresis gel (success on the transformation) and prepare some manganese ir order to complete our BG-11 medium.

4-9-2012 During the morning, we’ve managed to subculture two E.coli colonies from the plate that was transformed with pAM1573 (Susan Golden’s plasmid) , prepare more BG-11 (this time with manganese) which was at a 8.9 pH and gave us some brown precipitate and also preparing some marine broth to be ready when vibrio comes. Aside from this, we managed to identify on the registry part the BioBrick corresponding to the inverter and to the terminator.

We’ve spent this morning mainly in making two more minipreps for the Susan Golden’s plasmids. We’ve got results but they are not concluding so this evening we will repeat the electrophoresis in order to check out if this was what was wrong. Aside from this, we’ve spent the evening preparing Petri dishes with LB medium in order to grow some E.coli transformed nwith the inverter biobrick, the terminator biobrick and the LuxI gene, subculture this E.coli and the ones transformed with the pAM1573 plasmid (this last one only in order to check out possible mistakes) and prepare some Synechococcus medium (both BG-11 and plants fertilizer) with manganese.

During the morning, we spent all the time, checking out the e.coli culture state, preparing new meidum with antibiotics (kannamicin and ampiciline) on three Petri dishes, each of one corresponding to a different transformation (inverter, terminator and LuxI). Aside from this, we managed to In the evening, we check out that some growing has been observed in CsCB Coccus flasks that were left in the stove at a 35ºC (one with plants fertilizer and another with BG-11).

(Read with deep, profound, thunder-like voice): A new tragedy comes to our lab! Here comes the even more difficult! There you can see the self exploding ligase tube! Nobody know how did it happen, but the question is that happened. Apart from this, it has been a regular work day with all the minipreps from the inverter part, repeating some transformations (self correction: it has been a normal day). During the afternoon we began to do some Syechococcus CsCB experimental cultures with 200, 250 and 300 NaCl mM. Also we’ve designed the experiment for the sucrose measurement and prepared some minipreps for the inverter part which came from two different colonies and some digestions for the LuxI part, the pSB1C3 backbone plasmid, and the Terminator. It seems that the beginning of the academic course will limit our disponibility on the lab, so some days some of us won’t be able to come to the lab.

Week 10

Report from the morning: some BG-11 with NaCl autoclaved, and another electrophoresis done. The gel gives us some disturbing results, so at the evening we repeat the ligation and the gel- success this time! Aside from this, we’ve prepared more Petri dishes and done more digestions and ligations for the same BioBricks and plasmids from yesterday. In the evening, we checked out the experimental Synechococcus cultures, some transformations and some more minipreps.

11-9-2012 Preparing some 80% glycerol dilutions; it needs to be ready when we have our parts assembled. Apart from this, we did some minprep for the RBS, pAM1619 y pAM2314 plasmids- no problems with the gel, as some E.coli with these plasmids.

It’s not been a regular day at all; first of all, finally the Vibrio fischeri we’d demanded to be used as the light bulb of our biolamp is finally among us. Also, Victoria (Rocío’s flatmate has come to the lab to see us work and film some stuff for doing a promotional video). Aside from this, work seems to be growing day by day and we’re not sure that we will –self-correction: we were aware we wouldn’t have time to do everything we want from the very beginning-.

Nobody has been able to come today to the lab due to schedule’s incompatibility.

It has happened again- another day lost due to academic duties.

Week 11

Finally IPTG has arrived! Some good news in a busy day. Try to transform E.coli with the BBa_K084014 BioBrick, prepare some Petri dishes with antibiotics, BG-11 and some plants fertilizer in order to transform Coccus . Also the electrophoresis for the Susan Golden’s plasmid, and some Synechococcus CsCB subcultures have been done.

18-9-2012 Hallelujah! Finally the university has lend us one of their spectrophotometers for doing the Synechococcus growing measurements (a little note from the autors, the spectrophotometer we are using now is not the one which was taken from our lab and still stays in Calpe but one which is in the lab on the floor above us but we’ve been told not to use it until this moment).

Retaking an ancient tradition on our lab, here comes another epic fail: there are strong suspects that the vibrio bacteria we were supplied with was the wrong specie: it was nothing but V. cholerae (don’t worry it’s a non-pathogenic strain). The rutine remains the same in the lab despite of all this.

A little correction for yesterday’s entance: it’s not Vibrio cholerae but vibrio mediterranei. Despite of this, we’ve some good news: psbaI has arrived! Finally we will be able to do our biobrick! –better too late than never!- Minipreps, cultures absorbance measures and all the other stuff keep going on.

Due to practices and other features related to the academic course, no one has been able to come to the lab today- dammit.

Week 12

Biobrick finally assembled and sent! But the work is not over at all This seems to be like a 24 season finale. Everything seems to keep going on and on.

25-9-2012 Another fail strikes the lab! Unknown antibiotic resistant bacteria have appeared on our Petri dishes. Aside from this another trophy unlocked: do the sucrose measurements required for your experimental parts – just if our glucometer works.