Team:LCG-UNAM-Mexico:Journals:Nando's

Nando's lab journal

My main goal is described in these points.

1) I'm in charge of the assembly of the P4 vector. 2) I need to obtain the essential region of P4 sid 1 by pcr 3) I need to check for the characterization of p4 as an iGEM plasmid. 4) I need to ligate the P4sid1 plasmid to the BiteBack system and transform an E.coli C1-a strain with it. 5) then we can start checking for transduction of our system.

During the months of June and July, I was in charge of obtaining the phages to work with, T3, T7, P2 and P4. In order to accomplish this, I emailed the "Phage people". They are:

Dr. Richard Calendar.- Professor of Molecular Biology at UCB. He has gently handed phage p4 vir1, P2 vir1 and some strains to work with them: C-520, C-2423, C-1895 and C-331. Dr. Giuseppe (Joe) Bertani from CalTech. He kindly sent C1a, C-117, C1906. Dr. Mario Soberón and Sabino Pacheco from Instituto de Biotecnología UNAM (Institute for Biotechnology of the National Autonomous University of Mexico) have kindly given us a phage T7 lysate. Dr. Ian Molineux from the University of Texas gently sent us phage T3. P4 sid1.- obtained from ATCC

August 12, 09.

A very important piece of data is the phage titer you use, since you cannot know what properties the infection is developing if you don't know how many phages you threw into battle. So T3 and T7 are diluted in LB medium, which is a good buffer for them.

Another important point is the making of an agar or agarose mix called Top agarose. This Top agarose tops the normal agar plate (hence its name) with an agar in a lower concentration that promotes proper plaque expansion.

I started organizing the phage titration. You start from a phage lysate as a 1:1 sample, make a dilution of 1:9 of that one for a 1/10, and repeat for the new dilutions until you reach the desired dilution: from 10^-7 to 10^-13. This way you can correlate the number of plaques formed with the dilution of the stock. For example, if you only get one plaque in a 10^-10 dilution, you can state that your lysate has 10^10 plaque forming units (pfu) in the volume unit you started with.

Today, overnight standing cultures of C1a, BL-21 were left to grow at 37 degrees in order to assay for the viability of T3 tomorrow. One of C117 was placed for storing the supernatant next day.

August 13, 09

We also need to verify that T3 and T7 infect C1-a without any problem. Since the most suspicious phage in terms of viability is T3 because of all the time it waited in the border, i added .15 ul of the overnight cultures to M9LB for it to reach the log phase. when they reached log phase (3 hours), I added 50 ul of phage to each flask. Then I proceeded to place the cultures in Miguel's Incubators (and one in someone else's incubator).

After some time, the cultures were crystal clear, what qualitatively demonstrated the lysis of the culture by phage T3.

The C117 overnight culture was centrifuged at 13000 rpm to pellet the cells and store the supernatant.

Today, an overnight of C 2423 and c1a were put to grow.

Plan for tomorrow.- prepare the plaque assay for P4 sid1.

August 14, 2009

We need to verify the viability of P4 sid1, so many things have to be assessed.

As the legend tells, p4sid1 must:

a) not make plaques in C1-a or P4-containing bacterial lawns b)not make plaques qhere there's no bacteria b)not make plaques where there is no phage present c)make plaques in p2-containing bacterial lawns

When going to check the C1-a standing culture, it happens it didn't grow as an overnight culture is expected to, so all the experiments will be put off until later in the day. This means that I won't be able to see the plaques myself until monday since I'll party my birthday away in Mexico City.

August 17, 2009

Looking at the plaque assay results.- see august 14

1.NCLB- bacteria grow.- probably due to mistake in procedure. 2.NCC1a.- Negative control for C1a (contamination control).- grows well without plaques 3.C1aP4sid1 1.- some small plaques really far away from each other. probably because some P2 and coinfection in the ATCC lysate 4.-C1aP4sid1 2- some small plaques really far away from each other. probably because some P2 and coinfection in the ATCC lysate (a really curious effect) 5.-C2423 P4sid1 3- many well-defined plaques (internal control discards contamination) 6.-C2423 P4sid1 4- many well-defined plaques 7.-C2423 P4sid1 5- many well-defined plaques 8.-C2423 CN.- no plaques :D

Conclusion of the first part of the experiment.- P4 sid1 does work in the formation of plaques. P2 is actually produced in lysis, but in a really low number.

Today's plan.- the next part is to isolate colonies fro the non-plaque regions of 3 and 4 that could contain a plasmid-state or lysogenic P4.

August 18, 2009

Yesterday's experiments summary.-

The plates were arranged this way.

plate 1

colonies

1, 2, 3, 4, 5, 6.- different culture zones in the C1a plate which are not plaques . theoretically, may be one of these colonies could have been infected and transduced by P4, so I must assay for the presence of plaques. However, it comes into mind how i will assess the presence ot P4 inside of these bacteria by only means of plaque assays, given that P2 is a complete phage and could perform lysis on its own. This would mean to make a comparison between lysis of P2 alone and along with P4, and it's probably non-trivial to distinguish P4 plaques from P2 plaques. given all this, We should investigate about a better way to test with an internal control. Of course, the biobrick with a color signal could be excelent as an indicator, but we cannot wait for that. x,y,z.- zones of the bacterial plate generated that include plaques from P2 presence.

the presence of plaques reveals an interesting phenomenon; the P4 sid1 purchased stock has some P4 contamnation because it's a lysate. Dr. Bertani argues that it is of really low significance since one P2 is produced by 100 P4. The fact that you can see plaques involves that the P2 that come out are really efficient infectors of the neighbor cells, which happen to be in a P4-filled environment. If this werent the real way, the P2 phage wold be lost in the medium and, ocasionally, a cell would die, but that wouldn't necessarily produce a plaque in the agar.

E,F,G.- C2423 zones from August 14, 09 which were picked very carefully trying not to obtain any plaques. These work as pneative controls.

In other respects, we tried to infect bacteria in M9LB without calcium to see if that worked. The incubations were made with 100 ul of phage. Lysis didn't start, so we cannot combine the protocols for both phages and calcium is really important for P4 adsorption. I need to find out how to carry out the protocol with the materials we have here.

August 19, 2009.

I tried generating lysis in liquid medium by addition of the P2 vir 1 lysate to a series of overnight cultures which were added 70 ul P2 and 40 ul CaCl2. the cultures were:

bacteria expected No bacteria clear dish C1a being tested for P4 sid 1 x 2 plaques if infected with P4, no plaques if not C117 small plaques C2423 large plaques C1906 no plaques

August 21, 2009.

We have officially confirmed yeast contamination in the lab. Many petri plates have acquired a shiny white appearance. This is a big problem, since maybe many of the results expected before now are modified by the contamination. Undoubtedly, this will lag the development of the project. The next best option will be to produce streptomycin resistant strains of every strain present until this moment. The only thing left to be done to leave this round of experiments, since the negative results from the last experiment may be due to the lack of virulence of phage P2, is to probe the absence of P4 through colony PCR product analysis. The PCR was performed.

August 24, 2009.

The PCR results almost prove the absence of P4 thanks to a lack of PCR product amplification The only thing needed for a complete confirmation. Now, let's move on. We have asked Juan Pablo the technician to prepare streptomycin for growth of resistants. By the way, we asked him to prepare top agarose to go on with the phage plaque assays among other things, like M9LB medium and a 1M CaCl2 stock. Meanwhile, we left the bacteria growing in order to be ready for when the streptomycin will be used. The cultures left for tonight resistance round are as follows:

1.C1895 2C2423 3.C331 4.C1906 5.C520 6.C117a 7.C117b 8.C1a

Almost at the end of the day, we noticed that Juan Pablo was not told by anyone to prepare the streptomycin solution, but fortunately, we found him and he gave us some sp 100 solution, assuring that it was streptomycin. So we proceeded to prepare plates for tomorrow's assay.

August 25, 2009.

Early in the morning, I centrifugated and resuspended cells in 100 ul to plate and scan for resistants. At night, some resistants finally appeared, but we have to further prove that they are what we want them to be. We will discuss this with Miguel.

August 26, 2009.

C 1895.- small colony spots... c2423.- 2 or 3 colonies. let's check for morphology C331 nothing yet. let's wait a bit more. C1906.- really lots of colonies. maybe the antibiotic went wrong. C520.- too many colonies. check for resistance again. C117b.- let's wait for them to grow a bit more. C1a.- nothing clear. C117a.- practically nothing CN.- nothing

I planned to repeat the resistant-growth assay, but the cultures I left inan overday showed contamination in the negative control.

Late at night, I offered Paz my help in purifying PCR products. I commited a really stupid mistake when using the elution buffer before the wash buffer and treating the flowthrough as waste. I was completely ashamed of my error!!! Luckily, the PCR could be done again.

Laura had left some cultures she asked me to plate.

27 august, 2009.

Nothing resistant has grown yet. I'll wait a bit more. Laura's cultures show a bit different behaviour from yesterday's. C520 returned to normal (that means, yesterday's was contaminated). none of the others has obtained resistance yet.

Miguel finally told us what tests we can make for assessing a real resistance acquisition:

1.- to draw a resistant colony with a copper pole in a petri dish with the wildtype bacteria at its side. 2. assess for correlation of cfu with serial dilutions. (to assess that bacteria don't normally become resistant 3.

C1a

August 25, 2009.-

October 5, 2009.

The day started at almost 10:00, when an over(day) of c-1895 was put to grow to perform a lysis assay at night.

The plans for this day were.-

1.- centrifugate and test candidate lysates left on friday in order to prepare for the kit reactions, which need a starter 50 ml lysate. 2.- autoclave all the stuff needed and prepare supplemented P4 medium for the night assay. 3 prepare petri plates for testing the lysates.

I poured fractions of friday's lysate flask 1 and 2 and named them as follows.-

oct 2 1.1

       1.2
       2.1
       2.2

I also used two tubes from september 30's lysate which I wasn't able to centrifugate due to rotor space. the main idea of centrifugating is to clear out the cell debris from the lysate

and to further process the lysate until concentrating the phage or using it as the kit starting material. First I have to test for the presence of phage, so I will spend today concentrating the phage completely and testing for the infection of C2423, the indicator strand, in plaque production before and after concentrating the phage.

I also started a protocol to produce more lysate, this time without adding the EGTA, so the calcium is not chelated too soon. I verified lysis start in two flasks previously added bacteria incubated with phage about 2 hours after growth start. one of the flasks started lysing at A600 .66 and the other one at .4.

October 6, 2009.

We had an iGEM meeting where we discussed about some aspects of the presentation, wiki, poster and other stuff. I checked the A600 of the cultures I left last night and it wass about 0.65 in both, so lysis stopped at some point and the bacteria continued growing.

I'm really excited because the lambda DNA purification kit just arrived around 4 pm! so I'll get my DNA today and start with PCRs tomorrow (or maybe today).

In general terms, the kit has a standard protocol in manipulating phages. what changes is the anion exchange column. the protocol is a bit large, but time till getting purified DNA is a few hours : like half a day.

the steps are.- digest RNA and DNA, precipitate phage with PEG, lyse the phage and extract DNA, bind te DNA to a column, wash it and elute it.

From my perspective, carrying out the protocol was limiting in several respects. First, centrifugation on falcon tubes at 15000 g is not achievable on normal desktop centrifuge, so more power is needed. I need to ask Rosy how she does it. The other way around is to pour everything into eppendorf tubes and do it in a microcentrifuge, but that discards the 4 C temperature. as you use a narrow pipette tip, DNA may be shattered. Second, you need the tubes to contain exactly the same of everything in order to avoid balancing all the time (the other way is to have a balance...¬¬).

It seems that the best way to centrifuge the mixes is in a supercentrifuge and an ss-34 rotor.

October 7, 2009

Results from kit DNA isolation.- no DNA was seen in any of the fractions. There are many possible reasons, but the most likely ones is 1 .- the phage titer and 2.- the procedure bumps. everything will be clear in one more attempt and with a positive control.

Results of the day.- The application of the protocol improved dramatically. While yesterday I wasn't able to centrifugate at 15000xg, thanks to otto geiger and his supercentrifuge, now I could do it. the outcome was a real pellet of potassium dodecyl sulfate and proteins , which could be poured into a fresh tube with the loss of almost all the salt. Another thing I noticed last was that I could actually cold-centrifugate at >15000 x g with our own centrifuge and that RIGHT AFTER ELUTING, I MUST CONTINUE TO THE NEXT STEPS IN EPPENDORF TUBES for the DNA not to irreversibly sediment at the bottom of the big centrifuge tube.

I took small aliquots of the steps after denaturing in order to check for the quality of the procedure. I expect a small, but at least visible yield of DNA. I can start working with that.

All the plaque assays made in the morning had lots of phage, but maybe not as much as the Assays made on September 21 since the plaques were countable in most of them.

Gel electrophoresis.- The gel with the purification fractions is considered a complete success. Although the band seen is really thin and a bit difficult to see, the size is precisely the expected one. One thing to learn about the gel is that a high voltage and more time are required. Next time, I will try running the gel for 1:30 hrs and at 80 volts.

Given that result, I was motivated enough tu run a PCR for the essential and non-essential parts of P4. The essential part is a bit over 7 kb, and the non-essential part is around 3 kb. So the PCRs were performed this way: a reaction for each of the samples containing the template band, namely DNA1, which comes from centrifuge tube 1; DNA2, from centrifuge tube 2; Fraction 5 of the purification, the elution from the column, also had the band present.

one set of reactions attempts to amplifies P4 essential, and the other one, the non essential region. therefore, I prepared stocks for six reactions (3+3 with different primers each) and a negative control with the 7kb primers. I used lots of DNTPS so my reaction won't run out of them.

October 8, 2009.

The Gel electrophoresis of last night's PCR has practically failed with both primer sets. Though we cannot discard the  fact that the polymerase itself didn't work due to lack of positive controls, lanes of the essential region for sample DNA 1 shows two blurred spots at the size of the template  at the expected size of amplification (7kb). It's not overkill to try again changing some parameters, like the polymerase or the annealing time.

For today, I will make a restriction assay of my template in order to find out if it's the actual expected template. For this, I have decided to use the enzyme EcoRI, which has three restriction sites in the P4 chromosome. so the expected bands are as follows:

3 kb from site 1 to 2

Around 400 kb from site 2 to 3

around 7kb from 3 to 1

Partial restrictions.-

entire genome.- 11600 kb

1 to 3.- 3500 kb

2 to 1.- 7400 kb.- maybe undistinguishable

3 to 2.- 10000 kb... maybe difficult to see.

The restriction was completed to 75 ul with 15 ul of DNA when it was expected to be at 40. if it went wrong, I'll do it again.

The pcrs that were performed with RTth hot start were those of DNA1, DNA2 and fraction 5 (elution step) which happened to show the band of the genome.

if something goes wrong, we could blame the elution buffer's substances.

In the meantime, I'll try to obtain some concentrated DNA fro the eluted fraction. It needs to be added 1 volume of isopropanol and centrfugated for 30 min at 15000 x g.

The result of concentrating the phage DNA from the resting elution was completely successful. The band can be seen completely at the corresponding size and the concentration is considerably high. Restrictions and PCRs will be made with this new stock. at 9 o'clock, the restriction time will have ended.