Team:Tuebingen/Notebook/Journal/Weekly

Week 26

This is the final week in the lab! We got two more complete pRS plasmids with Pfet3-mOrange-Tadh1-pRS316 and Psuc2-mOrange-Tadh1-pRS316. Pfet3-mOrange-Tadh1-pRS316 was co-transformed with Padh1-mPR Xl-pRS313 in w303 wild type yeast. Fortunately, the transformation was successful, so we finally have a yeast strain for the next crucial step in characterization: to proove whether the membrane progestin receptor can influence the promoter activity of Pfet3 or not. The last microscope pictures of Psuc2-mOrange-pTUM100 and Panb1-mOrange-pTUM in the repressor deficient strains were taken. Furthermore we investigated the influence of changing the medium to SC-Ura (2% galactose). As expected Psuc2 activity depends on the carbohydrate source in the media!

iGEM 2013 ends with a lot of cleaning and sorting of all transformed yeast strains, plasmids, and E. coli glycerol stocks. See you at the Jamboree in Lyon!

Week 25

Finally! We found a good protocol for the fluorescence measurement in the Tecan-Reader! As expected, an 8 h long induction of Pfet-mOrange-pTUM100 gave good results. Assemblies of Padh1-mPR Xl-Tadh1-pRS313 and GAL-mig1-Tadh1-pRS313 were positive. In that way, we got our first fully complete pRS vectors! Unfortunately the restriction, ligation, and transformation of Pfet3-mOrange-Tadh1 and Psuc2-mOrange-Tadh1 in pRS313 had to be repeated due to problems with the vector. The ligation of Pfet3-mig1 in Tadh1-pSB failed. Sadly, there is not enough time left to assemble the complete system thus the ligation was not repeated.

Again this week: transformation of Panb1-mOrange-pTUM and Psuc2-mOrange-pTUM in the repressor deficient strains. Varying the time of the heat shock helped a lot! Panb1 required a heat shock of 90min to have good results compared to the 30min in standard operating protocol. We have also tansformed luc-pTUM104 in W303 wildtype yeast, but time is running out for the characterization of Luciferase...!

Week 24

Our first try to observe mOrange-fluorescence in the Tecan-Reader worked surprisingly well. Measurement in water is much better than in medium since YPD as well as synthetic complete drop out mediu have high self-fluorescence in the range of 500-600nm. We will need to perform a LIM induction over at least 8h for characterizing Pfet-mOrange. Overnight cultures in LIM did not grow at all and within shorter times (3-4h) no results were seen. Luckily, we finally found a terminator (also a Tadh1 derivative, BBa_K801012) which was already shipped with this year's distribution kit! Unfortunately, the part was neither marked as available nor as working. Why didn't we find this part earlier?! We lost almost three weeks waiting for Tadh1! Ligations of the most important 3A-Assemblies in Tadh-pSB (Padh-mPR X.l., GAL-Mig, Pfet-Mig, Pfet-mOrange, Psuc-mOrange) worked well.

Week 23

Thie week we were still desperately looking for a working and easily available yeast terminator because we had completed all useful assemblies of promotor and coding sequence. This week we started to introduce our new parts in pSB1C3 for shipping and archivation. We are planning to send seven parts to the registry, among them our membrane progestin receptors and the iron inducible Pfet3. Also, two complete inverter systems consisting of mig1+Psuc2 and rox1+Panb1! The first transformation of our plasmids in yeast worked, so we could mount our first yeast for microscopy. We were very pleased to see a bright signal for Padh1-mOrange-pTUM. Nevertheless the promotor actvity of Psuc2 was very low. Thus we prepared YPD plates of a mig1 deficient yeast strain hoping for a better signal there. We also started to prepare our own buffer for further yeast transformations!

Week 22

Tadh1 was promptly delivered and we smeared cells that were transformed with Tadh1 on one of our plates but got no colonies. A closer look in the registry revealed that there some issues with the part and we decided to order another one. While waiting for the terminator we started to ligate two parts without terminator in pRS316, namely Padh1-mOrange and Pfet3-mOrange. We also digested these parts with NdeI which degrades the pTUM100 backbone (has AMP resistance). A retriction digest revealed that pRS315 has one additional EcoRI restriction site which was not marked in the plasmid map.

We made a lot of media for our work in yeast: YPD, synthetic-complete drop-out media, SC-Ura and SC-Ura-His, low-iron medium (LIM), glucose (40%) and galactose. By choosing the pRS backbones for all parts wisely we only need three different SC dropout media!

Week 21

After another control restriction we had to face the bitter truth: luc had completely failed right from the start (i.e first PCRs). Thus we had to do everything that we thought we had accomlished again. This means: luc PCRs and DNA purfications. Last weeks 3A-Assemblies had mixed results (found by plasmid preparation): Psuc2-mOrange and Pfet3-mOrange were negative (however, we picked new colonies of these constructs and yielded better results); Padh2-mOrange, Pfet-rox1, Padh1-rox1, and Padh2-mPR Xl were positive; mig-pSB1C3 and GAL-pSB1C3 were positive, too. Another transformation of Tadh had negative results thus we decided to simply order this part from the iGEM HQ. A transformation of luc was negative (again...) thus we had to start again from the beginning (for the second time in one single week!).

Multiple 3A-Assemblies had positive results (cut with E/P then gel): Pfet3-mig1, Padh1-mig1, GAL-mig1, and GAL-rox1! Also, the ligation of Panb1 in pSB1C3 seemed to be successful.

Week 20

Another Panb1 PCR was successful! Thus we digested Panb1 with E/P and continued with a PCR-purification. Control restrictions of Padh1-mPR Xl-pTUM, mig1-pSB1C3, Panb1-pSB1C3, mOrange-pSB1C3 and RFP-pSB1C3 had mixed results. All parts that were ready at this point (Psuc2, Pfet3, Padh1, mPR Xl, mPR Dr, and mOrange) were prepared for 3A-Assembly by restriction digestion and eventually ligated in pTUM (mig1 and GAL (distribution-kit) were ligated in pSB1C3).

Week 19

After last weeks gelextractions we were able to ligate! We had Pfet3, Tadh1, mOrange (from distribution-kit), and mig1 ready for ligation in pSB1C3 and continued with transforming them. First gels of the resulting plasmid preparations confirmed that Pfet3 had finally positive results!

Week 18

This week, we mainly did gelextractions. We started the week off with gelextractions of Pfet3-pSB and mig1-pSB and continued with gelextractions of pSB E/P, pSB X/S, mig X/S, Pfet3 E/P, Panb1 E/P, and Tadh1 E/P. A very important step was our first 3A-Assembly of Padh1-mPR Xl in pTUM!

Week 17

We had some transformations going on and were able to pick Pfet3-pGEM, luc-pSB1C3, mPR Dr-pSB colonies. Therefore, we could continue with plasmid preparations of these parts. We also did preparative restrictions of mig1-pSB1C3, luc-pGEM, and luc-pSB1C3. Finally, we ligated mig1 in pSB and luc in pGEM, Psuc2 was ligated in pSB.

Week 16

Unfortunately, we found out that colony-PCRs do not work that well and result in wrong positives! In order to optimize our colony-PCR protocol (we had some issues with the amount of cells in the tube and the temperature) we made a gradient-PCR with eight RFP-pSB1C3 colonies. We did some colony-PCRs of mPR Dr and Padh1 using the new protocol and cut the plasmid preparations of the positve colony-PCR-colonies. The control restriction showed: Padh1 is positive!

Week 15

We transformed the ligations of of Week 14 into competent cells, performed a plasmid preparation on them and checked the results with colony-PCR and restriction. No postive results. Furthermore we ligated some more parts (Pfet3, Tadh1, Panb1) into pSB1C3 and made some gelextractions of Pfet3 and the plasmids pSB1C3 and pTUM100.

Week 14

We did digested (with E and E/P) all plasmid preparations of parts that seemed to be successful (until now) and had devastating results. We found out that we had had a lot of false positive results because of supercoiled plasmid with bands of the same size as our suspected parts. Only rox1-pSB1C3 and mPR Xl-pSB1C3 were confirmed, all other parts were back at start.

Week 13

This week, we did some transformations of rox1, Padh1, and Psuc2 in pSB1C3 and of Pfet3 and luc in pGEM. The result: rox1, Padh1, Pfet3, and luc had colonies! After controling them, we prepared glycerol-stocks of these constructs. However, Psuc2-pSB1C3 was negative. Since we were running low on money, we experimented with a protocol for plasmid preparations without kit and had some positive results.

Week 12

Again, last week's chemo-competent cells were useless because they had an endogenous AMP resistance - for whatever reason. Thus, we had to create competent cells once again --- this time: no resistance! We attempted a ligation of rox1 in pSB1C3 and ran gels of plasmid preparations of mPR Dr-pSB and mig1-pSB - at first, everything seemed to be positive. As we found out later the band was in fact the supercoiled plasmid!

Week 11

We checked last weeky transformations but had only negative results. In contrast, last week's transformation of mPR Xl in pSB1C3 was rather successful. Colonies of mig1-pSB1C3 and mPR Dr-pSB1C3 were prepared and digested with XbaI / PstI but were unsuccessful. Furthermore, we created more competent E. coli cells - that were AMP-resistant!

Week 10

After a depressing gelelectrophoresis we now know that last week's plasmid preparations that we have sent in for sequencing did not work out. Nevertheless, we did a colony-PCR, plasmid preparation and control restriction digest of rox1-pTUM100 and Panb1-pTUM100. A gelextraction of Tadh1 was successful and further refining of the gelextraction commenced. A transformation of Padh1 and Psuc2 in pTUM had no colonies and plasmid preparations of last week's transformations (Padh1-pTUM100 and Psuc2-pTUM100) had negative gels after a control restriction digest.We prepared restrictions of pTUM100, mig1, mPR Dr, and mPR Xl and attempted a ligation + transformation of mig1, mPR Dr, and mPR Xl, in pTUM100 and in pSB1C3.

Week 9

A new batch of chemo-competent cells had promising results. Our test- Transformation was a success, finally no colonies on the negative plate. However, PCR purification of Tadh1 did not work out very well due to its small size - Tadh1 might just flow through the columns without adsorbing. Restriction digests of pTUM103 and pTUM104 and pSB1C3 plasmid preparations seemed to work well thus we were able to ligate Padh1 and rox1 into pSB1C3 this week! Plasmid preparations of luc, Psuc2 and Padh1 had good results and were sent in for sequencing. A colony-PCR of Psuc2 and normal PCR of Tadh1 was successful thus further steps could be initiated. The restriction and transformation of Psuc2 and Padh1 in pTUM100 were unsuccessful. Still, luc, Psuc2, and Padh1 were sequenced. A transformation of rox1-pTUM100 and Panb1-pTUM100 yielded only few colonies - can't have everything in one week.

Week 8

Unfortunately, first exams reduced lab-work even further. We had a successful PCR of Panb1 and Tadh1 and attempted ligations of rox1 in pTUM (cut with X/S) and of Panb1 in pTUM (cut with E/P). We tried some Transformations on plates with ampicillin- aliquot, unfortunately there were still colonies on the negative plate.

Week 7

Control restrictions of Padh1 and Psuc2 with Xba/Spe and Eco/Pst restriction sites were accomplished, unfortunately there were no bands in the agarose gel. Likewise, we prepared some more PCRs of Panb1 and Tadh1 but could not identify any stains on the subsequent gel. We retried the agarose gel electrophoresis with a higher sample volume without achieving the desired results. Our transformed pTUM100-ligations of the parts mPR Dr, mPR Xl, mig1, and luc did not grow that bad, but we had a massive problem with colonies on our negative plates! Our first suspicion was, that the aliquot with the ampicillin-resistance did not tolerate the daily thaw-freeze-thaw-freeze procedure. Furthermore, we tried some PCR`s. However, we had neither product nor Primer-bands. In addition to this, we cut rox1 out of pGEM with Xba/Spe and purified it. Since many ligations and transformations resulted in empty vectors (or even worse: no vectors at all) we started experimenting with higher concentrations of chloramphenicol and ampiciline in our plates.

Week 6

Chemo-competent cells: next try. This time we actually did everything correct and had wonderfully working chemo-competent cells! Our first colony-PCRs of Padh1 and Psuc2 were successful - stains were at the correct positions in the initial gel. However, gelextractions of both cultures yielded empty vectors...

Week 5

This week, summer semester started thus time for lab-work decreased gradually.

We have created new chemo-competent cells since previous attempts failed due to insufficient cooling of cells. However, we had no luck again and had way too low trafo efficiencies with these cells. Even worse, Pfet3 PCRs still were annoyingly problematic for reasons we simply could not work out. Fortunately, we managed to complete a PCR of luc (hunderds of bp) and transformed luc in E. coli.

Week 4

Pfet3 PCRs remainded a huge problem - nothing we tried seemed to work out. Also, we have produced new chemo-competent cells and tried to test the pTUM-vectors by ligation with some of our parts. We also tried to amplify a full fragment of luc (after successful site-directed-mutagenesis). After many experiments we have finally found an ideal protocol for gelelectrophoreses that does not include expensive post-staining: we basically use more loading buffer and make the wells full to overflow!

Week 3

The first thing we did in this week, was a control restriction of last week's ligations. Unfortunately, not one single ligation was successful thus this whole step had to be repeated. Our work with luc (i.e. luc quick-change) continued, additional PCRs went well, and we made plans how to control luc's quality once both restriction sites were eliminated.

This week, we received a package containing pTUM yeast vectors from TU Munich's iGEM Team! We need the pTUM vectors from pTUM100 to pTUM104 in order to add missing BioBrick restriction sites to some of our parts.

Week 2

Further PCRs of Panb1 and Pfet3 were more promising but Pfet3 still made lots of problems because there were only very faint Pfet3 stains on our gels. We transformed pSB1C3 and RFP into E. coli and our first plasmid extractions of these parts went pretty smooth. We yielded pretty good amounts of DNA and after some restriction digests we were able to ligate Panb1 into pSB1C3!

We started out our iGEM project with several unsuccessful PCRs of Pfet3. We also tried restriction digests and ligations of Psuc2, rox1, mig1, and the mPRs into pSB1C3– without positive results. Unfortunately, we had massive problems (smiling marker) with staining our gelelectrophoresis gels thus had to start off our project with trouble-shooting. After some experiments we came to the conclusion that post-staining would be an expensive way to solve our gel problems. Due to better gels we could prove the successful PCRs of Panb1 and Psuc2 and promptly started ligation and transformation of these parts. Initial transformations of mig1 were unsuccessful. However, luc PCRs went very well and we already had highest hopes for this part. Unfortunately, we found two illegal restriction sites (EcoRI and XbaI) inside of luc thus we had to come up with something special: site-directed-mutagenesis (quick-change) using 3 pairs of primers and Pfu-polymerase. Restricitons of Padh1, Psuc2, rox1, mig1, mPR Dr, mPR Xl and RFP (our initial reporter) went rather well but due to missing negative controls in the control gel further steps were not possible this week.