Team:UNITN-Trento/Notebook
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Revision as of 07:55, 10 September 2012
Our Lab Book
Filter by:
- Filter by:
- Black Crust CysE
- Black Crust CysDes
- Terminators
Week 1 (05/21 – 05/27)
We start to meet in the lab. We have been assigned a big lab in Malga, the
building that hosts all the teaching labs. It’s the biggest one.
We’re starting to design the constructs and primers and prepare LB
medium for culture. We also make a list of all we have at our disposal and
what we need to order.
Week 2 (05/28 – 06/03)
During the weekend I and Guzz go statue hunting: we stop by a few
marble/statues stores and ask for a few samples. We find kind people who
let us take what we want, so we now have what we need to test the
parts we will hopefully build.
I continue to research on the statues project, while developing a Python
script together with Guzz to detect illegal sites from sequences returning their position if they exist, or adding prefix and suffix instead.
I continued with the purification of many plasmids and PCR products, in particular LacI from pET-21 plasmid and AraCpBAD from E. coli genome and from the Registry.
Week 3 (06/04 – 06/10)
The primers to PCR-extract CysE from the E. coli genome arrive, so I can
now start with it.
My first electrophoresis, obviously, fails. I let it run too much. We now
know that these gels run much faster than the one used during exercitations.
In my next attempt I manage to see the product on the gel (gel photo). I proceed with the restriction of CysE and pSB1C3 (the submission vector) with E and P, and then ligation and transformation in Nova Blue cells.
The transformation succeeds, giving both red and white colonies. The
screening of a white one confirms my first construct, and our first Part! HIGHLIGHT: 06.08 Please Welcome K731000 (gel photo)!
I selected a couple of possible terminators to test from the registry (B0010 and B0017), and I purified some of these colonies.
I continued with the purification of many overnight cultures and PCR products, in particular LacI from pET–21 plasmid (which would have become one of my parts) and AraCpBAD from E. coli genome and from the Registry.
The starting was fantastic: Giaco brought a cake!
We designed some primers, tried to extract some parts from registry, such as some standard vectors.
Starting designing the terminator construct: we decided to use the Roberta Lentini’s plasmid RL0024, with the fluorescent proteins Cherry and Venus. This will allow us to calculate terminator’s efficiency as a ratio between the peak intensity of Venus and Cherry, avoiding problems of various variables that could influence raw measure.
To submit it we must mutate two illegal sites, XbaI and PstI, and insert prefix-suffix in the linker between the two proteins (take a look at our final construct).
To take measurements we’ll use the E. coli strain BL21pLysS, an expression strain with both polymerases. A familiarizing week also with protocols and various tools.
Week 4 (06/11 – 06/17)
I can now apply a mutagenesis PCR to CysE to obtain M256I CysE. The gel confirms thar PCR worked (gel photo), which is then ligated into a digested pSB1C3 and transformed again in Nova Blue cells. I’m at the Univesity Olimpics when I have to take the inoculi from the thermoshaker, so Andrea kindly does it for me.
The screening, again, confirms my second Part K731010 (gel photo). At this point I have the building blocks to make the my Composite Parts, using araCpBad as promoter, which is made by Ross.
I and Ross then decide to start with araCpBad: we digest CysE with E and S, araCpBad with X and P and pSB1C3 with E and P. Ligation and transformation into Nova follow.
I went on constructing my first BioBrick, which is K731300: it consists of LacI-LacIq into pSB1C3. Everything went right, and on 13th June I could announce the birth of my first construct, after attending the endless run of a giant 2% agarose gel (I waited for about three hours…!) (gel photo).
In the rest of the week I kept on isolating and purifying some low-copy plasmids that could have been useful in the future.
Transformation of some Roberta’s plasmids: just Venus (RL009), just Cherry (RL007) and our template RL0024.
I also tried my first mutagenesis, prefix-suffix insertion, but it failed…we got a multiple band gel, meaning that the primer didn’t annealed properly. After some attempts we decided to order another primer suffix-reverse, because we thought that one annealed non-specifically. This new primer was designed for a slightly different template: RL0030A, that’s identical to RL0024A except for a 30bp longer linker between the two proteins’ genes.
Amplification of some plasmid backbones from registry.
Week 5 (06/18 – 06/24)
The construct me and Ross made last week is purified but not yet quantified and screened; anyway we want
to send it for sequencing before the Post Office closes (along with CysE
and M256I CysE) or the samples will be stuck during the weekend (and we don’t
want that!) We estimate a concentration of about and blindfoldingly
prepare the samples. Cristina rushes and between traffic and parking is in
front of the Post Office 5 minutes before closure. In the meantime, sadly,
the construct is NOT confirmed by screening. Doh.
I prepared all the samples for sequencing, and I continued purifying various plasmids and helped out other people in the lab. And I also studied for the exam of Developmental Biology.
This week Giaco and Ross went to CiBIO (research center) to try FACS for the first time: they took RL007, RL009, RL0024 in BL21, and got expected results.
I failed another insertion PCR; also with the new primer on RL0030A the gel kept on showing multiple bands.
This week Giaco and Ross had a honey weekend here in lab, all alone…îScience weekend has begunî [battle journal citation]. I don’t know exactly what happened, but anyway Giaco got the first mutation: the PstI site (gel photo).
Week 6 (06/25 – 07/01)
We start over, this time inserting CysE after araCpBad in pSB1C3, cutting
the vector (which contains araCpBad) with S and P and then insert (CysE)
with X and P. Last time we used the registry protocol; This time we used higher quantities of DNA. For the insert we used PCR products rather than a plasmid.
The screening gives a variety of results. I realize we’re very unlucky, again: screening with E and P, which gives vector and insert bands, results in fragments of the same length. Cristina finds there’s a BamHI site near the end of araCpBad, so I can screen with BamHI and PstI to confirm the cloning (gel photo). I also make a PCR using Prefix and Suffix primers, just to be sure (gel photo). Both are good, meaning I have my third Part.
This week I also begin to work with the mutated version of the composite
part: araCpBad-M256I CysE. I try with a modified version of the 3A method, the one I didn’t manage to do well with Ross last time. This time, instead of simply digesting and ligating the biobrick , I PCR out CysE* and araCpBad and then digested them separately.Afterward I have ligated them into pSB1C3 in one shot.
The screening of the transformation (with many many red colonies) is not
good, again. The 3A assembly seems my nemesis.
This week our gene synthesized by Genscript has arrived! I will be working all day long from now. From now on I will have to sleep in the lab.
I immediately started to try to amplify the gene by PCR, and after 12 attempts I finally succeeded in it (gel photo). In the weekend I purified and digested all the parts that I would have used to create my new parts, K731400 and K731450.
On the 27th June I will start working on the constructs assigned to me. Up until now, I've helped put others.
I will build and characterize our new lac operator-containing plasmids. I will then pass the information I gathered to Andrea and help her testing her parts.
These are the constucts I will create:
Name Description Backbone
K731500 lacI_lacIq_pTAC_lacO pSB1C3
K731510 lacI_lacIq_pTAC_lacO pSB4K5
K731520 lacI_lacIq_pTAC_lacO_GFP pSB1C3
K731530 lacI_lacIq_pTAC_lacO_GFP pSB4K5
The gene we ordered is pTAC_lacO_RBS_CysDes into pUC57 and flanked by prefix and suffix. Since I need to extract the lac operator alone, along with Cristina I designed the primer lacoperator_rv, the reverse complement of lacO followed by the suffix reverse complement.
Since the ordered gene needs to be amplified, I’ve transformed it into DH5α.
I’ve had some troubles amplifying the 139bp construct, since the designed primer annealed with better affinity to the 30bp-long suffix. The results were two bands, one more intense corresponding to the whole ordered construct (unwanted), and one less intense of the desired length. After a couple of tries with different conditions and buffers, I’ve been able to selectively create and amplify the wanted construct (gel photo). The key is extension time: shortening it to 10s prevented the amplification of the long construct.
Numerous attempts of amplifying the ordered gene by both me and Andrea resulted in low yields; it is strange, since pUC57 is an extremely high-copy plasmid…
This week we tried to insert T7 in pSB1C3, but from the sequencing we found that it didn’t work.
We also tried to extract the right band of the RL0024*(we designed with a star each mutation made on the construct)+prefix-suffix from the gel, but the attempt failed: the concentration of the final sample was almost nothing.
Discovered that the prefix-fwd annealed also at the end of Venus (the two proteins have N-term homology), thus we ordered ANOTHER primer, this time the prefix-fwd one…
When it arrived, I tried again this insertion, both with RL0024 and 30 and sent for sequencing (gel photo).
Giaco obtained the second mutation, the XbaI site (gel photo).
Week 7 (07/02 – 07/08)
What I can do now is transferring araCpBad-CysE from the submission
vector (which is high copy) to a medium copy vector; it will be needed
when I have to characterize it (it seems light years far now). We choose
pSB3C5, digest it with E and P along with the PCR made to screen the
construct in pSB1C3.
I carry on with the ligation and transformation in DH5a, that the screening
with both EcoRI and PstI and BamHI and PstI confirms (gel photo). And another Part goes to the stock.
For M256I CysE, I decide to go on with 3 different approaches
at the same time: inserting M256I CysE after araCpBad in its vector (this approach worked with the WT version), making new overnight cultures from the failed 3A Method of the previous week (you never know) and making a mutagenesis PCR on the confirmed WT Composite Part. Ironically, the one that doesn’t fail the screening is the second one (the one I made with the least hope). LOL.
I make a PCR from the construct (in pSB1C3) with BioBrick primers (gel photo) and digest with E and P, to insert it into pSB3C5 digested with the same
enzymes.
I did not start the week in the best way, because I won the “King of the lab” title already from Monday… But even if I made some mistakes at the beginning of the week I managed to create my two new constructs by the end of the week! Here it is the gel that confirms that I won my battle:
(gel photo)
In the weekend I started the preparation of MOPS medium, that we are going to use in the next weeks to start to characterize our constructs; and I also began to make another new construct, K731600.
K731500 has been created. I use Andrea’s part K731300 and the lacOp part amplified by PCR from the gene we received from Genescript. It's hard to confirm it by electrophoresis since Andrea's and mine constructs differ by 0.15kbp.
Sequencing confirmed the correct mutation of both illegal sites.
We remade the insertion PCR once again, using the new primer, but then we discovered that I made ANOTHER mistake: I forgot to exclude from the primer itself the EcoRI site that was in the linker between the two proteins… We had to order ANOTHER primer to be used in a insertion-deletion-point mutation PCR!!!
We also tried to digest the construct with EcoRI and then ligate it, to see if maybe in this way we could eliminate the double EcoRI site.
Week 8 (07/09 – 07/15)
To finish with the mutated construct I transform it into DH5a and confirm it
by screening (gel photo). I choose 3 colonies to send for sequencing, one of which has the vector band lower than the others (as a negative control).
And now it’s time to start thinking about the characterization of the construct I
made. Ross is currently testing the promoter expressing GFP, so his
results will serve as a guideline for my experiments.
Since I can’t actually see if the protein is expressed in real time, I use
Gibson’s Assembly to attach a sfGFP at the end of M256I CysE directly in the
medium copy vector. I’ll transfer it in the submission one afterwards, to let
me test it first. The PCR fails.
However, this doesn’t mean I can’t start to characterize: I transform araCpBad-CysE WT (I’ll call it 1025 from now on) in NEB10b, our chosen cell line for expression. I make my first real characterization experiment: I grow cells overnight in LB, dilute them 1:50 and, when they reach an O.D. of about 0.6, split and transfer them in minimal medium (MOPS) containing respectively Glycerol and Glucose as carbon source. At O.D. 0.8 I split them again, and induce
one culture with arabinose (0.5mM). I take a sample from each culture before induction, after 4h of growth, and after 8h. Every hour after induction I measure the O.D, to get growth.
This was the week of the medium: MOPS had to be ready as soon as possible to start with the characterization of our constructs. And this fun work was given to me! …I spent about three days weighting salts and filtering solutions drop by drop…
Anyway, in the meantime I also prepared the Molecular Biology exam and then had the first failure with my new construct K731600: the ligation products did not survive to the transformation, neither one good colony grew up. So I prepared a new digestion: but only at the end of the week I realized I have used an “unreliable” plasmid: the final concentration resulted horrible, so I needed to start again from the beginning amplifying a new pSB1C3 and digest it again.
This week will be remembered also as “the week of the first contamination”: plates of all of us started to have weird halos, and from now on the contamination started to be a fellow traveler of us.
I confirmed my construct (K731500) by screening in a 2% agarose gel. (gel photo?) I'm waiting for the sequencing results. I've been working on K731510, I will have the results next week.
We played a little with the fluorimeter, measuring BL21 coltures with just Venus or Cherry, our template with both proteins and another construct that has them in the opposite order (RL0020). The coltures were inducted with IPTG 0.1mM and we analysed fluorescence at different times of induction. We also tried to left them O/N in the refrigerator and we found out that the fluorescence greatly increased.
We repeated this kind of experiments several times, testing also RL0024 with the double mutation of the illegal sites.
One day Mansy came to see how it was going on and to make fun of us a little (that is a kind of relaxing hobby for him), and we discussed together what could be the best parameters for the future measures:
Excitation | Emission | |
---|---|---|
Cherry | 587nm | 615nm |
Venus | 485nm | 528nm |
Then we began to test some others features of the protocol: we tried to sonicate the samples, and took an enormous amount of data at different induction times and different lapse times after sonication. We repeated each experiment several times and with different colonies from the transformation time. We were completely adsorbed by this work but organize and analyse all that data was far worse than take them…
We defined the final parameters for fluorescence measurements:
* 2–3h induction 0.5mM IPTG
* brief sonication
* proteases inhibitor treatment
* O/N in refrigerator
Of course we also tried another PCR to insert prefix-suffix and of course it failed.
Week 9 (07/16 – 07/22)
The characterization obviously continues. I make changes in the protocol to find the optimal method; I try, for instance, to grow directly in MOPS A (i.e. glycerol) overnight as Keasling does. After a few tries I find that the original approach (growing overnight in LB and then changing the medium before induction) works best for me (my cells).
The sequencing results for araCpBad-M256I CysE (which, again, I’m gonna call 1035 from now on) arrived, and confirm one colony with an high confidence (I sent 3, but something went wrong with the primers so we only have half of the sequencing, I send those two back).
I can now transform 1035 in NEB10b and characterize it too. This is the version that should work best on the statues, thanks to its mutation.
And this is the time when the Tragedy, as we call it, occurs. In a moment of confusion with lots to do, I see Guzz is transforming. I give him my cells and miniprep of the 1035 confirmed colony. And what Cristina feared most happened: Guzz was transforming ligations. When we transform ligations we transform all the product, so we put cells in the ligation eppie. He does that with my (only) miniprep of 1035 confirmed colony. Which means that I have none of it now. Please note I’m not blaming Guzz for this, my part was my responsibility: my part, my mistake.
I decide to transform it and hope the cells will survive from the DNA excess and let me miniprep my plasmid back. I am now to transform another colony in NEB10b, I choose one of the two that was half sequenced and look promising.
In the shadow of all this, a change in the conditions of the Gibson’s PCR (2°C higher for the annealing step and a 30s longer initial denaturation) proves successful (gel photo). Along with Andrea, who’s making the same thing with her CysDes, I use the Master Mix for Gibson’s Assembly and transform it in the good old DH5a. The screening, again, is kind of ambiguous. It seems, though, that the GFP is not there.
Coming back to characterization, I have to say this phase of the project is very tiring. I’ve to stay until late hours to get to 8h of induction. I also add serial dilutions to the mess: at 4h and 8h I plate serial dilutions of the cultures to estimate the number of cells, as measuring O.D is not a direct method.
Growth curves results are accumulating, I now have triplicates and have to
find the time to analyze them.
By the end of the week minipreps of the Tragedy are screened, confirmed
and sent for sequencing. I try with one that looks good on the gel in
NEB10b.
This was very tiring. I had too many things to do, and as they were not enough I had also to prepare the Biochemistry exam. But as it usually happens in situations when you are tired a lot of mistakes are made, starting from my usual unlucky construct K731600: this time the digestion did not work, and I had to transform twice before screening. I also started to prepare a new construct (K731660) with a different low-copy plasmid, pSB6A1, but the digestion did not work.
Fortunately I was lucky with another new construct, K731480: it contains LacI-CysDes+sfGFP into pSB1C3 (PCR gel photo 1 and 2), and I constructed this BioBrick following the Gibson assembly protocol. I have screend 6 colonies, only one was correct and its overnight culture was fluorescent too! Anyway the screening seemed ok (gel photo), we had just to wait for the sequencing results.
(gel photo)
During this week I also started to characterize my construct K731450, or better, I should have started to characterize this construct: as I said at the beginning I was very tired, and I forgot to add the antibiotic to the medium; so I threw away the culture, I will start again next week. After the Biochemistry exam of course, and after my first weekend of relax and freedom.
I've confirmed by screening the construct I created the previous week. Also, sequencing results of K731500 are OK.
I’ve tried to create K731520 and K731530, but the screening is a total disaster.
I tried to characterize anyway those constructs, by choosing and amplifying the most promising colonies. While I was able to measure IPTG-dependent levels of fluorescence for K731530, the other one did not seem to contain GFP gene. Just half a success.
I’ve been told that the chosen approach doesn’t work. Others tried it even before I started my job and failed… if only I knew.
We’ve also been in Trieste to meet the only other italian team. It has been fun, but I had to stay in the lab until 3AM after that.
I will amplify K731530 by PCR and try to insert it into pSB1C3 to obtain K731520 – a safe approach…
This week we mutated again RL** with the new primer using a three steps Phusion program, ligated and transformed it. The screening digestion was good (gel photo)
and sent for sequencing some samples.
Wednesday and Thursday I stayed at home to study for an exam and Giaco had a lot of fun without me: he screened the effective presence of the terminators B0010–14–15–17 in the parts extracted from registry. He found out that there was nothing in the parts B0010 and B0015 but the vector pSB1C3. He tried also to amplify them by PCR but the gel showed nothing once again. On the other hand, he confirmed B0014 and 17.
He got through some difficulties but finally he made t7terminator in pSB1C3 (gel photo). Last time, when I tried, seemed all positive but the sequencing revealed just empty plasmids, so we were quite concerned about it…cross fingers…
Immediately we set up a screening digestion using EcoRI and XhoI (that has a site after Venus) but the gel showed just the band of the entire vector! Noooooo!
We were stunned and desperate, and we didnít believed that: we made another screening digestion in the same way and left it O/N.
Even the morning after the gel presented us the deep nothing… We spent the rest of the weekend thinking if it was worth to go on.
Week 10 (07/23 – 07/29)
One realization makes my Monday bright: in the chaos of the Tradegy we didn’t realize that the colony sent for sequencing was the negative control: the vector seemed shorter, and since we can’t see it on the sequencing, I decide to drop the Tragedy colony. Haha.
Sequencing results are back to complete the ones we only had half, telling me the colony I’m using is good.
I have now enough data from growth curves and serial dilutions, so it’s time to see if the proteins are actually working, beside being produced (as growth curves and dilutions suggest.. we can’t tell it for sure until we make a protein gel or have the Gibson’s construct to test).
What I’m doing is a Ninhydrin test on the samples after 4h, 8h or overnight
to see if there’s Cysteine in the medium, the final product of CysE activity.
We find an old paper by Gaitonde et al. describing the protocol. In this test ninhydrin is solubilized in a solution of glacial acetic acid and concentrated hydrochloride (it takes 20 minutes even at max speed vortex). You add the reagent to a cell sample + glacial acetic acid at 1:1:1 ratio. When heated at 90 degrees Celsius the color changes to a pink/purple in about 2 minutes; the more intense the more Cysteine there is.
My first try is after 4h of induction, using 1025, 1035 and empty NEB10b. After 10 minutes only one sample is intensely pink.
From our understanding of the pathway, only 1035 in MOPS A (with glycerol) should be induced. We make this assumption and joy. But, turns out it was 1025 in MOPS B (with Glucose). We begin to ponder, and come to the conclusion that our assumption was obviously wrong. The induction in glycerol or the absence of glucose alone provides the cell the ability to highly express the protein, which may impair growth (as growth curves suggest). The higher OD in glucose, in
contrast, could be the cause of the pink color of cultures in it: the basal transcription or the one not favored by glucose presence might be sufficient to produce enough Cys. I should investigate more and retry the test on the cells left to grow overnight.
The results the day after seem confirming our hypothesis: the lighter pink is the induced culture in 1035, the other follow.
Another attempt to get the Gibson’s to work: I try to add DpnI to the PCR products already made last time, then purify and use the Master Mix and so on as before.
Contamination continues to persecute us: this is the plates’ turn. And of course I threw away my ligation product of K731660 again; as it was not a priority I decided to stop trying to construct this part, maybe I could have started from the beginning in the future if I have had time.
A good news from the sequencing arrived: my construct K731480 with GFP is right! But I had the confirmation just looking at the plate, actually…
(photo)
Is this enough fluorescent, isn’t it?
Anyway also bad news came from sequencing results, and they involved K731600 of course… I sent two samples to sequence, one contained an unknown gene and the other had CysE and not CysDes as insert…! I can't explain how it could have happened. It remains the fact that this construct is cursed, and I had to start again from the beginning.
This week I finally started to characterize my constructs, and I began by making growth curves, serial dilutions and fluorescence measurements of K731480. I did every measure twice, and I tried with two different carbon sources: glycerol (that is in MOPS A) and glucose (in MOPS B). I won’t show you every chart and data I have, you can find them into the file “Characterization of CysDes”.
… which almost failed.
I screened twelve colonies instead of the usual ten. The 11th and 12th seemed successful. (gel photo) Also, bacterial pellet was fluorescent at the transilluminator, so I’m pretty confident. While I wait for sequencing results, I will try to characterize the constructs.
K731530 has been fully characterized. In order to do so, I’ve done duplicates in a single day, for a total of 144 measurements. It has been harsh, but necessary.
On Monday our advisors came back and even Mansy didnít believed that sequencing. He suggested we set up another (yes, another one) screening digestion; we run the gel and….magically the bands appeared!! (gel photo)
We`ve got it, finally!!! We failed so many times making it but eventually we succeeded!
Such a good birthday present!
In the rest of the week we amplified the correct construct and screened several colonies from the T7terminator-pSB1C3 ligation; they seemed all positive (gel photo), so we sent some for sequencing.
Amplifying the right Cherry-Venus construct we had, ironically the bad luck kept to persecute us and that construct: I failed several times to miniprep the overnight culture, and when Giacomo had mercy and helped me, I spoiled also his ones!!!
Anyway we had lot of fun even in failing, and this success put us in a good mood.
We often found that this was the better solution, but not this time: we got multiple band product (and the major wasn’t the right one). Thus we made another PCR, two steps: this time the gel was much better (gel photo) and we transformedÖ and sent for sequencing.
We also made two terminator’s insertions: T7teminator in the T7promoter plasmid (gel photo) and B0010, extracted by PCR from Bba_E0840 (PCR gel photo), into the same vector (gel photo). The more shocking thing of this week, anyway, is that we and all the team tided and cleaned up all the lab!
Week 11 (07/30 – 08/05)
After a couple of qualitative tries of the ninhydrin assay, it’s time to go quantitative: after the reaction has finished I jump to the spectrophotometer and read the spectrum of the samples. Cysteine has a characteristic peak at 560nm. The results correspond to the intensity of the pink.
However, repetitions of the test on different days are not so consistent, as
the peaks of the same sample type are at different heights. The ratio and
“order” is maintained, though.
This is also true while building a standard curve for Cysteine, made by
providing a known quantity of it in the samples: peaks have doubled heights the second time, confirming this test is not so reliable. It still lets me know that the protein is doing its work, luckily. Even if it’s not exactly what we expected.
One time I “steal” 1mL from Andrea’s overnight culture. She’s in charge of
CysDes, the second protein of our pathway. She’s also in the process of
characterizing it, so she always has cultures ready too. I use her version of
the protein in a vector providing Chloramphenicol resistance, in order to be able to inoculate cells along with mine, as all my construct are in Chloramphenicol too. I induce the mix at the proper OD with both arabinose and IPTG (she has the LacI promoter). The ninhydrin test gives me promising results: samples are of the same color as control, which probably means Cysteine is actively being degraded by CysDes. We need to try a more systematic analysis.
Ross, who’s almost finished his characterization, can start to support me in
my efforts. It’s kind of relieving, I was struggling to keep up with everything.
He takes over my Gibson’s construct, completing the screening and
confirming the construct. He’ll also transfer it in pSB1C3 and characterize it.
I dedicated this week to characterize my constructs K731400 and K731450 as I did for k731480 (without fluorimetric measurements, of course); and I also started AGAIN to construct the cursed BioBrick K731600. I will win this battle!
But even this time something went wrong, because contamination was still waiting for us; and so I threw away the first ligation product. Then I made one mistake plating onto the wrong antibiotic, throwing away the second ligation product. This forced me to be in the lab at 5am the next Monday, to recover lost time and not to slow all the work of others, whose samples should have been sent in the morning of 08/06 together with mine. This time I was very confident, I felt it was the right time; but my up early revealed totally useless, as you will read.
K731520 has been fully characterized. I’ve been able to reduce the delay by doubling the work load on the last days, and with this my job is done.
I’ve spent a lovely Sunday at Caldonazzo’s lake with my girlfriend. It has been my first free day in three weeks: up to saturday, it has been 23 consecutive days of work.
We tested the restriction enzyme activity digesting our terminator platform with XhoI and E, P, S, X.
This time the digestion was made in a total volume of 20µl to avoid influence of glycerol concentration.
Also decided to look for a better way to run gels of such little fragments: finally we tried with a 1.5X agarose gel, with a little more EtBr and TBE instead of TAE (gel photo). Wow!
We were also too confident: we amplified and made an insertion on a Ptac plasmid not yet sequenced… Friday the results arrived and was really puzzling: we took a mutated sample but 6/8 were correct…such a bad luck!
We started right away with the correct colony and made all the remaining clonings:
* pSB1C3 with B0010, B0015 (gel photo)
* T7 terminator platform with B0015 (gel photo)
* Ptac terminator platform with T7, B0010, B0015 (gel photo)
During the whole cloning phase Roberta and Laura gave us a great help: also this time we had B0015 (registry 2011) from them, since with couldn’t extract it from the 2012 reg.
Sincerely, I think I had a bug part in this mess…
We threw away antibiotics, plates, cells, LB, transformation buffer, glycerol…everything!
It was really hard to eradicate it, but finally we won and get back to normal…
(T7 and B0010 in platform were confirmed by sequencing)
Week 12 (08/06 – 08/12)
My tests continue with even more likeable results. Our interpretation seems to be correct. Ross also confirms my protein is being expressed.
It’s time to start to amplify the confirmed Parts, giving priority to those in the Submission vector. We set August 15th as the deadline to finish reamplifying.
To avoid confusion and mistakes, I transform a Part a day, so I always have to do overnight cultures or minipreps of different constructs on any day of this this week. By Friday I have one left to do, which I leave for next week.
To use a relevant expression, this week we set a milestone of our project: we start to put cells on marble samples. I put induced and not induced cells of 1025, 1035 and empty grown for 4h in both MOPS A and B on a marble slide and cover it with a gel matrix Ross optimized. We leave it overnight. I also immerse half of a marble piece in induced 1035 cultures.
Truth being told, for me it’s a bad week. My Mac is sick. I need to go home to know what’s wrong, but I think the hard drive failed or the RAM got corrupted. In the meantime Sheref lends me an old Mac from his lab.
Too bad I can’t install nor Lightroom nor Espresso, the software I need for
photos and to build the website. I still have my iPad, so I can easily keep tracking my experiments and write this very journal.
On Thursday I have a meeting with Cristina and Sheref to update them on
my results and decide if I need to gather more data to put on the presentation for the Jamboree. It’s not the case, we decide. I should focus on experiments on statues from now on. We also make an outline of what will be my thesis.
The same evening I jot down a more detailed outline with OmniOutliner
and I am happy with the result. Next month is gonna be a tough ride.
My presence in the lab at 4.45am in the morning of Monday was totally useless, because even this time my attempt of construction of K731600 miserably failed. I cannot stand this part anymore.
But I did not give up, and on Wednesday I started again from the beginning: it is a personal challenge, I will make that construct by the end of the summer. This time I made every possible screening during the cloning, and when it was time for screening on Sunday…. The gel after the digestion was illegible, maybe the agarose had some problems and the samples did not run as usual; so I had to digest again and wait until Monday and use a new type of agarose for screening. What’s wrong with this construct??
In the meantime I did some tests to verify and quantify the good working of my gene CysDes, that produced H2S starting from cysteine. I tried the tests suggested by Keasling in his work (the methylene blue test) and a test suggested by the iGEM Yale team 2010 (the BCS test). I obtained good results. However I will have to repeat the test: the plate from which my coltures came from was a bit old, and the gene did not work at the maximum of the possibilities. For this reason I will repeat both the tests next week.
Test suggested by Yale:
In this test you add CuSO4 in the colture medium. If your bacteria produce H2S the Cu binds to sulphide and precipitate forming covellite; you can measure how much Cu is remained in the solution adding BCS (bathocuproinedisulfonic acid), which immediately reacts with Cu and turns orange. Then you record your results measuring the absorbance of your samples at 483nm: less the Cu in the solution more your bacteria worked and produce H2S. In parallel you can measure the OD of your cultures and see how it changes in relation at the production of H2S.
In my case I obtained a partial positive result: how I was expecting in the not induced culture there was no change in the concentration of Cu, meaning that H2S was not being produced; in the induced culture it is clear a decrease of the concentration of Cu between 3 and 4 hours, and it is positive. Actually the decrease maybe should be clearer throughout the whole time of incubation, and for this reason the next week I will try again the same experiment with a fresh culture of bacteria.
Test suggested by Keasling:
In this test you verify the production of H2S observing the turn of color of your solution upon addition of XX: if there is production of H2S it turns immediately from yellow to green/blue.
Even in this case my results were partially: one colture worked perfectly (CysDes-GFP induced), while the other colture (CysDes K) worked a bit worst: you can see a little difference in the color between the induced and not induced (the induced is little more green), but the induced one is too yellow: I think that this is always due to the too old plate. I will repeat the test next week with a freshly transformed plate.
I exposed the gathered data to the group.
Starting from this week, I will work on Biosafety and Future Application sections. It’s not a light task, but it’s something new, so I’m happy about it. I don’t want to hear about cloning anymore.
I will also work on modeling.
Along with Ross, I also started to characterize co-cultures.
Eventually I reach also this week, that’s the present one.
On Monday we did nothing but jokes with the team; Tuesday we started to measure some terminators. We measure the fluorescence of T7-platform, B0010-platform and platform and compare all the data. We did a preparatory step to find the best way to measure also absolute values of peaks, as we want to see if the terminator’s presence influence Cherry expression.
The results were amazing: the coli terminator seemed to work much better than the T7, and both terminators seemed to stabilize Cherry, in addition to reduce Venus. This behaviour wasn’t expected and we were really surprised and pleased of that.
Also the sequencing results arrived, and confirmed Ptacplatform with t7 and B0010 terminators. Also pSB1C3 with the same two terminators was confirmed. No construct with B0015 was right, but luckily they are less important.
Week 13 (08/13 – 08/15)
This week was short, because from 16th to 19th we decided to have a break and go on holiday! So I worked only on Monday and Tuesday before cleaning the lab on Wednesday and taking the train to return to Romagna.
In these two days I had time to fail again with my cursed construct: I screened 6 samples by digestion and also by PCR, but all the agarose gels I made were awful and it seemed that ligation did not work (again). Cristina wisely decided not to send anything to sequence before holiday, we were not sure about the goodness of my samples; it would be better to make new overnight cultures on Tuesday and screen them once returned to work in the lab on next week. And this is what I did, fortunately.
In the meantime on Monday our friend Damiano came back to the lab to help me in measuring the H2S concentration of my colonies with his portable gas-chromatography. We had excellent results: empty cells did not produce H2S as expected while cells with CysDes produced a concentration of about 18ppm. This is a good result, because we demonstrated that my construct works but it does not produce a concentration too high of H2S that could be dangerous. Excellent!
I’ve characterized co-transformations.
I’ve done a few tests on marble pieces.
With Ross, I’ve started building an acid rain simulator which should be able to reproduce the process of black crust formation.
In this week we tested the terminators’ behaviour with Ptac. The results were quite surprising: although the terminator efficiency calculated with Venus’ raw peaks seems similar to that found with T7 promoter, the stabilizing effect on Cherry was considerably lower.
This discover was really puzzling for us: it seemed that the stabilizing effect depends on the polymerase used and we had no idea how to explain this phenomena. Maybe this is due to the polymerase processivity or quickness??
We planned to do that next week with the help of Fabio Chizzolini in the Mansy Lab.
With a really freudian disattention we mistook the antibiotic in the colture for the second repeat of the Ptac measures: we started our holidays a little bit before!!! Yes, we are going on holiday for 4 days!!!
We spent the last day talking about project’s logo and name.
Week 14 (08/20 – 08/26)
Come back to work. It was hard after seeing sea, sand and friends for just a couple of days, but an hard job was waiting for us. We had a long meeting at the beginning of the week to sum up all the things we still had to do, and returned home a bit shocked after realizing the amount of work that was still waiting for us! But on Monday we had already completed a long and debated discussion: we had the name of the project, CRUST AWAY! And now we only missed the logo, but Nadia was working hard to realize it for us.
This was another important week for me, except for the name of the project: I completed my construct K731600! After screening the 6 new colonies I inoculated before holidays the PCR revealed the presence of my insert in the g colony
(gel photo)!
Actually even the i colony seemed to have my insert, so I sent for sequencing both the colonies for sure. On Friday morning I was at the post office to send the samples with a great great great hope in my pocket.
Meantime I repeated the methylene blue test for three or four times to test new samples and collected good data to show. I definitively verify that this test does not work with the lysates of the co-coltures CysE+CysDes; one of the hypothesis is that the cysteine production by CysE is too low to be used by CysDes to produce detectable quantities of H2S. Anyway I repeated the test only with my coltures of CysDes and empty cells with different concentrations of cysteine, I obtained very good results.
This week we took another repeat of the Ptac measurements (in reality they were two but I’ve lost the data of the second one).
Moreover, we did the first in vitro reaction, using the Ptac just-linker-construct. The reaction was done with PURExpress kit and a spectrophotometer different from the one always used. We took the fluorescence data for 6h, then the day after we took another measurement at the usual instrument. We had a great help from Fabio Chizzolini and Silvia, from the Mansy lab, doing this in vitro reaction.