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Notebook

WetLab

• Basic Flip Flop

  • Week 1

  • Week 2

  • Week 3

  • Week 4

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9

• Improving Flip Flop

  • Week 1

  • Week 2

  • Week 3

  • Week 4

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9

• Epigenetic Flip Flop

  • Week 1 (July, 4-8)

    Initial contact with the lab. First of all, it is necessary to amplify some vectors that will be used in the construction of the modules, Pnmt1(41X) and Purg1, as well as two constructions that have been gently sent from the Dr. Attila Becskei from the University of Zurich. On the other hand, I have designed some primers that will be used later.

    There are still lots of things to do.

  • Week 2

    Short week. The order of synthesized part tetR-CSD has just arrived to the Lab, so I can start to build up the compaction proteins modules. First of all, I have had to transform the plasmids into E. coli. Also, I made a PCR to amplify swi6 from the genome of Schizosaccharomyces pombe, and sir3, from a plasmid. The objective was to get enough DNA of these parts and, by using appropriate restriction sites that were introduced into the designed primes, start assembling the fusion proteins for this module. Next week we will see if the experiments have worked out as expected.

  • Week 3

    This week I have started to assemble the DNA fragments that will be used for the compaction protein modules, so firstly, I have had to made sure that all its parts were available. I set up an inocula of the E. coli previously transformed with the tetR-CSD plasmid and made a miniprep. At the same time, I checked the results of the PCR of sir3 and swi6. I have just been lucky with the last one.

    I have repeated the PCR to get sir3 from the plasmid, using different conditions: I modified the initial concentration of the DNA template and reduced the annealing temperature), but the result was again negative. Then, I have launched a new strategy and cut the plasmid containing sir3 gene with restriction endonucleases to purify the entire gene and use it late as template for a new PCR. This strategy also allows me to check that the plasmid was the right one.

    In the meantime, I have made sequential cuts of tetR-CSD, pPnmt1(41X) and swi6, all the components of the compaction module, apart from the slippery sir3. However, we have got some bad news. The rest of the synthesized parts that we had order will be delayed at least four weeks. Desperation!!! Let's do it old-style way, amplifying the parts with primers and assembling them with DNA ligase!

  • Week 4

    I began this week trying to clone into the pREP41X vector the parts of two compaction modules (tetR-CSD, tetR-Swi6). I transformed E. coli DH5α with the ligation products. The following day, I used a new method for screening recombinant clones, colony cracking, but I didn't obtain positive results. So, I tried with minipreps and digestions, but results were negatives too. I followed the same protocol, but digestions didn't work.

    I changed again the PCR conditions to get sir3 and made a gradient PCR, but it didn't work either. To make sure that the template was the correct one, I made analytic cuts of it, with different restriction enzymes. After two tries, results were conclusive. What's wrong? New primers.

    Apart from that, obtained Tadh1-tetOn (ADH1 terminator with a variable number of tetR operator sites repetitions). For doing that PCR conditions had to be optimized (annealing temperature, template concentration, extension time, …).

  • Week 5

    I digested again tetR-CSD with the appropriate restriction enzymes, XhoI and XmaI, but and electrophoresis with the digestion products demonstrated that there wasn't cuts. Analysis of tetR-CSD DNA sequence showed that restriction sites were lost. A site-directed mutagenesis was order to recover restriction sites.

    I made analytic digestions of pPR013 and pPR074, obtaining coherent results.

    I also amplified and purified Swi6 using an optimized PCR reaction, in order to use it when needed. Amplification of sir3 with new primers gave no results. So, I need to optimize the PCR conditions.

    At last, we received another synthesized part: tetO2. I transformed DH5α E. coli with this part and the following day I inoculated isolated colonies to make minipreps.

    In the meantime, I began to construct the Purg-GFP-Tadh1 module, a positive control of the designed module containing the tetO flanking sequences. This construction will be integrated into the genome of S. pombe at the leu1 locus by homologous recombination.

    In other hand, I ligated the fragment containing the Tadh1-tetOn obtained by PCR into pGEM-T vector and transformed it in E.coli. Colonies will be screened after the holiday break.

  • Week 6

    When I came back from holidays, I found the rest of synthesis parts (GFP·tetO4 and tetR·CSD) over my table. I used the standard protocol to transform them into E coli.

    I made digestions of tetO2·Tadh1 and Purg with BglII restriction enzyme.

  • Week 7

    Colonies of transformation plates were quite strange. I set up inocula of them and repeated the transformation protocol. Some of the inocula didn't grow up, maybe because of selection of satellite colonies in ampicillin plates. I made minipreps and digestions of them, to get tetR·CSD (XhoI/XmaI), tetR (XhoI/BamHI) and GFP·tetO4 (PacI/AscI). I also digested pREP41X plasmid with XhoI and XmaI, in order to ligate tetR·CSD and tetR+Swi6 between pnmt41X promoter and nmt1 terminator. However, the result of the ligation was not positive possibly because the plasmid was not properly digested.

    I made an electrophoresis and I tried to purify TAdh1·tetO2 and Purg from agarose gel, but TAdh1·tetO2 fragment migrates with bromophenol, so I had to repeat this process again. Then, I proceeded to ligate them o/n while keeping my fingers crossed, and transformed ligation product in DH5α the following day. There appeared colonies in spread petri plates. Colonies cracking didn't work, so I set up inocula to do minipreps next day. After an immeasurable colonies screening, I obtained two positive clones (19 and 35), Tadh1·tetO2·Purg is ready.

    Digestion of Tadh1·tetO2·Purg with AscI and PacI is made to introduce the next component of this module, GFP·Tadh1·tetO4. I had the same problem: plasmid was not totally digested and control religation plate was plenty of colonies. Lesson learned: if you don't give enough time to a reaction, you'll be wasting your time.

  • Week 8

  • Week 9

• MiniTn7BB (David Caballero)

  • Week 1 (July, 4-8)

    First week in the lab!

    During the last few months we have been studying and designing a Tn7-based system to deliver BioBricks into bacterial chromosomes. Now it is time to perform the ideas and experiments we drew on paper.

    We ordered the synthesis of a complete basic mini-Tn7 to avoid site-directed mutagenesis, cloning and other intermediate processes. Unfortunately, our order is delayed (22 days by now). So, this week I tried to prepare all the necessary strains and vectors in advance. Anyway, this is my first week in Fernan’s lab at CABD, so I am mostly getting familiar with the location of materials and equipment.

    These were my main tasks this week:

    1. Prepare pUC18-SfiI vector for mini-Tn7 insertion. Our mini-Tn7 was designed flanked by SfiI target sites. We plan to use a pUC18 vector, replicative in Enterobacteriaceae to maintain the mini-Tn7. I located the vector in the lab strain collection, set up an inoculum, purified the plasmid DNA, measured the concentration with the Nanodrop and digested with SfiI.

    2. Prepare pUC18R6K-SfiI vector for mini-Tn7 insertion. The goal is to obtain a vector with an R6K replication origin, which is non-replicative in pir- strains to host the mini-Tn7 constructs. We requested a pUC18R6K-mini-Tn7T-Km vector from Herbert Schweizer's lab in Colorado State University. We will use this as a template to amplify vector sequences adding SfiI restriction sites. I had problems to get amplification in the PCR reaction (twice), so I did analytic digestions with different restriction enzymes (four different cuts). The restriction patterns are not as expected. I may have used the wrong template (maybe the tube was mislabeled). I made new mini-preps, double-checking that the strain was the right one. Next week I will try to do this again with the new miniprep DNA.

    3. Prepare large amounts of the vectors necessaries to transpose the mini-Tn7. we will need fairly large amounts of pTNS2 (containing the Tn7 transposase complex), pUC18R6K-mini-Tn7T-Km (positive control), pBBR1MCS5 (negative control) to electroporate into the recipient strains for the transposons. I streaked the strains on selective plates, set up inocula, purified plasmid DNA and measured the concentration of the DNA, which was generally lower than expected. Also I did some checking of the pTNS2 plasmids with restriction enzymes.

    4. Finally I started a protocol to make electro-competent cells of Escherichia coli and Salmonella typhimurium, two of the four strains we will use to characterize the behavior of mini-Tn7.

  • Week 2 (July, 11-15)

  Our order of DNA synthesis has not arrived yet. It is a month behind-schedule by now and we are getting anxious about this delay of the DNA synthesis service. I know I must use my time in the lab despite not having all the required materials, so I made a list of all the experiments I can run without the mini-Tn7. Actually It is a long list with a fair amount of work to do. I set priorities and start from the top!

  1. First of all, I need a suicide vector I can use as the mini-Tn7 delivery system. Similarly to last week, the new minipreps of pUC18R6K-mini-Tn7T-Km yielded no PCR product and the restriction pattern with five different enzyme combinations was again not what we expected. Despite our efforts, it is evident that the plasmid we received was not pUC18R6K-mini-Tn7T-Km as published. After contacting the Schweizer lab about this problem, they informed us that they had sent us a different, although similar plasmid, but unfortunately it is not suitable for our purposes. Instead, restriction analysis confirmed that the transposase-delivery plasmid pTNS2 is based on pUC18R6KT and contains an oriT for conjugative mobilization, so we decided to use this as a template. Unfortunately, our SfiI-bearing primers are not suitable for this plasmid, so we designed new primers to amplify the pUC18R6KT backbone from pTNS2.

  2. The last set of mini-preps I did to purify pTNS2, pUC18R6KT-mini-Tn7T-Km (now discarded) and pBBR1-MCS5 had very low yield. I repeated the process and obtained somewhat more concentrated, but still low-yield preparations.

  3. This week I attempted to prepare several batches of electro-competent cells that I will need for the project. The strains are E. coli DH5α-λpir, E. coli MC4100, and S. typhimurium LT2. The former will be used for cloning into the R6K plasmids, and the other two will be used as hosts for insertion of the miniTn7 transposons. The protocol requires growth of large (500 ml) cultures OD600=0.5, followed by repeated washes with sterile, cold water. Cells are concentrated, allocated in 40 l samples and stored at -80°C. It sounds easy, but I had to repeat the process three times due to beginner's mistakes.

  4. I did mini-preps of two vectors expressing the Flp recombinase: pFLP2, a replicative vector which is lost spontaneously at high frecuency in Pseudomonas strains; and pCP20, which has a termosensitive replication origin that allows replication at 30°C but not at 37°C. This last vector will be used in E. coli and S. typhimurium.

  • Week 3 (July, 18-22)

  This was a less stressful week. We asked the DNA synthesis company about the 1-month delay and they claimed they had problems with the synthesis and still need two more weeks to have it ready. We are getting desperate about this! Still, I keep on preparing material in advance of the arrival of the long-awaited mini-Tn7.

  1. Checking the electro-competent cells I made the week before. I electroporated my electro-competent cells with a pUC19 vector to measure their transformation frequency but I hardly obtained any colonies. Being suspicious that the pUC19 preparation may be bad, I repeated the transformation with a more recent preparation of pBluescript SK(+). I obtained approximately 107 transformants/g for the three strains. This is an acceptable frequency for MC4100 and LT2, but we need a really high transformation frequency (over 109 transformants/g) in order to perform cloning experiments with DH5α-λpir. We decided to prepare a new batch of DH5α-λpir electro-competent cells.

  2. The mini-Tn7 was designed with a Gm resistance cassette flanked by selected restriction sites to facilitate exchange for other resistance cassettes. To construct mini-Tn7 variants with different markers, I designed primers to amplify kanamycin and chloramphenicol resistance cassettes from the vectors pSB4K5 and pSB1C3. Also, I did mini-preps of these vectors.

  3. Another thing we would like to do with the mini-Tn7 is to generate a handful of functionally useful mini-Tn7 vectors. The idea is to insert into the BioBrick cloning site of the mini-Tn7 useful BioBricks, like reporters for gene expression measurement, inducible promoters and constitutively expressed fluorescent and bioluminiscent labels. We selected the following BioBricks:

     • Promoter measurement cassettes: BBa_K093005 (RBS+mRFP1), BBa_I732094 (LacZα+GFP-AAV).
     • Inducible promoters: BBa_I0500 (pBad/araC), BBa_I732083 (pLacIQ+RBS+tetR+STOP+ptetR).
     • Strain labels: BBa_I13521 (ptet+mRFP), BBa_K325909 (pBad+Lux).

     This week I transformed these BioBricks in DH5α and prepared the corresponding plasmids.

  4. In order to test the site-specific insertion of the mini-Tn7 transposons in our strains, I designed primers to amplify from the Tn7R end to the glmS gene. I took this opportunity to train my self in doing sequence comparison and analysis using common tools such as Clustal and Blast. I searched for a conserved motif to make one primer suitable for all four bacterial strains, but the conserved sequences were not long enough. In consequence, I designed two different glmS of primers, one for E. coli and S. typhimurium and another one for P. aeruginosa and P putida.

  5. attTn7, the insertion site of the Tn7 transposon, is very well conserved in Gram-negative bacteria. However, if you want to use our mini-Tn7 based BioBrick delivery system in an organism without the attTn7, don’t worry, we plan to create a "portable" attTn7 for you. To this end, I designed primers to amplify the attTn7 site in the E. coli genome.

  Not having a better task to do, as you can see, this was primer design week for me

  • Week 4 (July, 25-29)

  Back to a hard-working week. As my time in the lab passes I realize that I can perform more experiments in less time, which does not always mean to get better results. Wednesday was an incredible day: I did more of the molecular biology technics I already know, and our mini-Tn7 synthesis order arrived. I exploded with enthusiasm. These were my main tasks this week:

  1. Checking DH5α-λpir electro-competent cells. I electroporated the previously prepared cells with a pBluescript SK(+) vector and I got approximately 2.5 10 ^{transformants/μg. It was not the expected >10 transformants/μg, but it is an acceptable frequency even for cloning experiments.}

  2. Not amplifying the pUC18R6KT vector from pTNS2. This week I made a huge effort to obtain a suicide vector to deliver the mini-Tn7, but finally I did not get it. I performed at least one PCR reaction with this aim every day, changing conditions (polymerase, nucleotide concentration, DNA concentration, different primer preparations, even already made PCR mix) and PCR cycle (temperature and time of steps). In the last try I performed a gradient PCR to determine the optimum annealing temperature with a PCR mix kit where there was not much that could do wrong, but even then I did not get any amplification. Then we thought that the SfiI restriction site we added to the primers could be avoiding the annealing, so we designed new primers where some nucleotides of the SfiI restriction site were part of the complementary region of the primers and others without the SfiI tail to have a positive control for the amplification.

  3. Mini-prepping. I repeatedly had problems to get highly concentrated plasmid preparations. I used mini-prep kits, so I did not know what my mistake was. I tried different kits and Fernan supervised my work one day, but the error was not obvious. By the end of the week, when one of the solutions of the kit ran out and I had to change it, my mini-preps started to rise their concentration.

  4. Not amplifying resistance cassettes. I tried to amplify kanamycin and chloramphenicol resistance cassettes from pSB4K5 and pSB1C3 vectors, but after three PCR reactions changing conditions I did not get an useful PCR product.

  5. Preparing BioBricks to insert them into mini-Tn7. I set up inocula, did mini-preps of the plasmids and digested them with EcoRI and PstI restriction enzymes. The next step would be isolate from gel, but the concentration was too low to use the fragments for cloning; so I repeated the digestions with high amounts of DNA, but the results were similar. I need mini-preps with higher DNA concentrations.

  6. ynthetizing a portable attTn7. I performed two PCR reactions changing dNTPs concentration and annealing temperature to amplify the insertion site of the Tn7 transposon from the E. coli chromosome, but I did not get any amplification. I preformed this process as a colony PCR (picking a colony to get the DNA template), so next week I will try to amplify the desire region of the E. coli chromosome by purifying the genomic DNA first.

  7. Working on our own-designed mini-Tn7. I performed the first necessary steps to do with a synthesis order: resuspending, transformation in DH5α, plating, setting up inocula and mini-prepping (this was one of the mini-preps of the last day so I got a highly concentrated preparation).

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9

DryLab

• Modeling

  • Week 1

  • Week 2

  • Week 3

  • Week 4

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9

• Simulation

  • Week 1

  • Week 2

  • Week 3

  • Week 4

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9

• Bioinformatics

  • Week 1

  • Week 2

  • Week 3

  • Week 4

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9

• BioBrick

  • Week 1

  • Week 2

  • Week 3

  • Week 4

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9

• Wiki

  • Week 1

  • Week 2

  • Week 3

  • Week 4

  • Week 5

  • Week 6

  • Week 7

  • Week 8

  • Week 9