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Team:UPO-Sevilla/Project/Notebook
From 2011.igem.org
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 DH5a 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 DH5a 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 DH5a 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:
- 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.
- 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.
- 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.
- 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
Week 3
Week 4
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
Sponsors
