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Team:Freiburg/Notebook/Toolkit
From 2012.igem.org
The way to the Toolkit
2.7-8.7Extension PCR of all the already synthesized direpeats on one plate
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Different colors indicate the six different primer pairs that were used during the PCR reaction
The Direpeats CC,CG and GC (yellow) were still missing from the synthesis company
We checked the Extension PCR with 4µl of 12 random direpeats and a additional template missing control which was sitting in F1.
We digested AA and AC with Pst1 and Xba1 the digested direpeats were ligated in the psB1C3 vektor and we tranformed 50 µl DH10B Ecoli. The transformed Ecoli were put on plates and incubated over night. No colonies could be detectet and the experiment was redone but without any results
We figured out the problem of the last week, the primers we used for extensions were missing a piece at the end which made it impossible for the restriction enzymes to cut. We didn't wanted to order new primers because they are quite expensive and it took the synthesis company three weeks the last time and we were already late. As a new strategy we were trying to ligate our extensions into pJet blunt end vectors and start the restriction digest from this pJet plasmids.
We tried our new strategy with AA and AC and ligated them according to the pJet clonig vector protocol. We transformed DH10B bacteria with the ligated vectors and put them on plates.
The grown colonies were picked for colony PCR and grown in liqud LB amp media.
We mini preped the grown picks and used the preped plasmids for a digest with Pst1 and Xba1
The digests were ligated again in psB1C3 and used for transformating DH10B. Again we tried to plate them out but like in the last time we didn't get any colonies.
We tried the experiment again and were able to get a few colonies. We did colony PCR and were able to prove that the size of the insert was like expected.
We started to do the pJet strategy on the rest of our 96 direpeats to get them all to the level were they are inside the pJet vector and already stocked in DH10B
We spend the whole week ligating, transformating and picking colonies for colony pcr and liquid stocks.
We tried the digest and ligation in the iGEM vector and got some colonies. The results of a colony PCR showed the right size. We started growing them in liqud LB and minipreped them as our first finished Biobricks
After all the work from last week got some wierd results from colony PCR on some Direpeats and started to sequence some of our finished Biobricks. Sadly we could see that what we thought were direpeats was nothing like the direpeats. After some sequence alining we could identify the problem as our extension primers. Because of the more or less matching sequences of the restriction sites our extension primers formed not just dimers but trimers which created fragments with the almost exact same lenght of the expected direpeat. We checked some of our finsihed biobricks and found a overwhelming number of them with just these primer inserted. With this information all of our finished Biobricks aswell as the finsihed direpeats in pJet vector could be thrown away because from colony PCR alone we could not be sure to have the right fragement.
As a conclusion we need to get a new strategy for creating our biobricks
We had a Labmeeting because of the problem from last week. After discussing the problem we stopped the pJet strategy. As a new strategy we came up with a somewhat wierd approch. The problem we are facing is produced by the missing of basepairs directly after the outer restriction sites. To solve this we want to order a new pair of primers that fits on every end of the six different extension primers which is not a problem because they are all the same at 3' and 5'. With this new short primerpair we extend our already extended direpeats so that the restriction enzymes can now bind again and cut at the restrictions sites. This is much cheaper and also faster, because we can get such primers in one or two days instead of the 3 weeks for the long extension primers.
We got the new Primers (so called ExEx Primers for extension of an extension) we started some testings to make a proof of concept experiment. After picking some clones we could se taht our strategy worked but it didn't work as good as expected
We ran several rows of experiments to get the perfect set up for the PCR and the ligation afterwards. After some failures mostly on the ligation with the psb1c3 vector we were able to create a complete protocol of how to get from direpeat to biobrick.
After sequencing a first proof of concept experiment with this new protocol we now having our first complete six biobricks AG1-6.
A new problem came up, at the beginning of our project the synthesis products of our direpeats were cloned in pJet vectors and then stored in Ecoli Cryostocks. These stocks were grown in LB media and minipreped to get the direpeats. Because we had all this pJet problems we sequenced all of the sixteen cryostocks and checked if the direpats were really in there. Sadly they werent in there.
Good for us we still had some of the original synthesis product left and could start a amplification PCR from them.
The last month of the project started already and we are now completely out of plan. We started the toolbox from zero and are now day and night at the lab. We are making some progress but slowly the protocol we are using is made so that there is no chance we are getting any false-positives. The digestion is done overnight and we are running all the digests on a gel to cut out the positive lanes. All of this takes time but we are now getting biobricks.
First 35 biobricks are finsihed and sequenced, another 30 are in line waiting to be digested. We are a little short on hands in the lab but we are working to get it done. The homepage is also in the making, and we are positive to take atleast the toolbox to the jamboree
Now we are at 70 biobricks and rising, the homepage is getting done we implemented dropdown menues and a slidshow with pictures. With a little bit of luck we can finish the toolbox this week.
No luck to report, the last direpeats TA is messing with our brains, it just doesnt want to be a biobrick
