Team:Bielefeld-Germany/Labjournal

From 2012.igem.org

(Difference between revisions)
Line 401: Line 401:
-
$("#tab ul").tabs("#labsite > div", {effect: 'fade', fadeOutSpeed: 400});
+
$("#nav ul").tabs("#labsite > div", {effect: 'fade', fadeOutSpeed: 400});
});
});
</script>
</script>

Revision as of 14:12, 23 September 2012

Prologue

Zusammenfassung

=Prologue= ==Starting the team== Beginning in january and february members of the former iGEM team from Bielefeld started seminars to inform interested students about synthetic biology, iGEM and the past Bielefeld projects. In March the final 2012 iGEM Bielefeld team was formed of 15 students and weekly meetings began. Our team was established and it was time to find a suitable project. ==Find a project== The first weekly meeting were more like big group brainstorming and we discussed idea, which in some cases were totally different from each other. Everyone had to inform about ideas of others so that, in the end, we all could discuss together. First project ideas were: * the detection of multiresistent pathogens * communication between bacteria and fungi using quorum sensing * a bacterial hand warmer * a possibility to detect and destroy mold fungus * something about spontaneous combustion of hay bale * an enzyme dispenser For most of the ideas little information was available. For example spontaneous combustion of hay bales is probably a combination of the metabolisms of different microorganisms and fungus. After some reports in media and press about the environmental effects of steroid hormones, we decided to go for hormones. From the beginning our aim was not to detect but to degrade hormones. We found several possible ways for degradation as there are the hydrolysis of estradiol-derivates with sufatases and glucoronidases. But we thought the best way to degrade steroid hormones would be with the use of laccases. Laccases have the ability to radicalize aromatic rings and can therefore be used to degrade or polymerize a broad range of substances, such as steroid hormones, special insecticides, polycyclic aromatic carbohydrates and aromatic acids. In nature laccases are often used for degradation or polymerisation of lignin or pigments. ==Molding together to a team== After we found our project idea we decided to have a get-to-know-weekend with some presentations about iGEM, important methods and ideas for human practices. We also held presentations about other possible iGEM projects to extend our horizon, as there were: ''e.g.'' RNA aptamers and magnetotactic bacteria. But the most important part of this weekend was the growing as a team. We realized that we all had one summer to work together, have fun together and most important to stand up together as a team. ==Find the right== Now it was time to organize the work and find a suitable task for everyone. In a developing team a lot of different jobs have to be done, ''e.g.'': * finding sponsors * communication with the public * human practices * wiki- and homepage-design * modelling * a forum for exchange of information * a joker, who entertains the team and lifts the mood And finally lab work began, feel free to follow us in our weekly labjournal and have a look how our labwork, results and of course problems and their solutions, evolved.

Summary of Week 1

We began our time in the lab with the cultivation of ''Xanthomonas campestris'' B100 and ''E. coli'' BL21(DE3) to isolate the genomic DNA to do PCRs and purify the desired laccase ORFs. In Order to do this we at first had to designed the PCR-primers. We decided, that the forward primers had to included the prefix, the T7 promotor, the RBS and the first 20 bases of the gene of interest and the reverse primers should consist of the last 20 bases of the gene of interest, a His-tag, followed by two stop codons and the suffix. Furthermore we started making preparations for the [https://2012.igem.org/Team:Bielefeld-Germany/Human_Practices/StudentAcademy Student Academy]. The Students Academy is a week-lasting summer school (9th to 13th of July) we got the chance to take part in organizing it. It is distinguished with a lot of presentations and lessons for pupils, but also guided experiments they had to do by themselves. Therefore the general idea for our experimental part was to give the students an understanding of the principle methods in biotechnology and synthetic biology by using fluorescent proteins. So the first step was to searched the Parts Registry for two plasmids with different fluorescent proteins and antibiotic resistances, respectively.

==Week 1 (04/30 - 05/06/12)== __TOC__ ''' weekly seminar:''' * Do we want to order strains of ''Trametes versicolor'' and ''Trametes villosa''? * Gathering information about signal sequences in yeast * Decision to create a database, so that we can easily number and inscribe our lab results * Decision to arrange a summer school for pupils in their last year before the final exams * Discussion about how to meet a member of the german ''Bundestag'' (the german parliament) === Monday April 30th === * '''Team Student Academy:''' We got the chance to organize one part of the first school academy “synthetic biology/ biotechnology” at the CeBiTec of University Bielefeld by arranging experiments for the pupils and by presenting us and the iGEM competition. For the experimental part our general idea was to give them an understanding of principle methods in biotechnology / synthetic biology by using fluorescent proteins. We planned the following experiments: ** Plasmid isolation of RFP/GFP from a liquid culture. ** Transformation of a plasmid mixture consisting of two different fluorescent proteins (e.g. RFP and GFP) and different antibiotic resistances into ''E.coli'' KRX. It will be plated out on LB agar plates without antibiotics and on plates containing one of the two antibiotics, which are present on the plasmids. This way we can demonstrate the effect of antibiotics as selective pressure. * '''Team Bacterial Laccases:''' :*Before our lab time started we sent requests for different plasmids to working groups, which have already worked with laccases we are interested in. Sadly just one working group responded to us. We got answer for a vector with the laccase-ORF [http://www.ncbi.nlm.nih.gov/protein/194015788 CotA] from ''Bacillus pumilus ATCC7061'' and a ampicillin resistance from the Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials in Switzerland. They promised to send us the plasmid pBpL6. [http://www.biomedcentral.com/1472-6750/11/9 More information...] :*In a [http://www.ncbi.nlm.nih.gov/pubmed/21790191 paper] we found a research group who worked with the laccase [http://www.ncbi.nlm.nih.gov/protein/21230052 CopA] from ''Xanthomonas pv. campestris ATCC33913''. Luckily the sequence of this laccase is the same in ''Xanthomonas campestris pv. campestris B100'' which we got from a working group at our university. The same thing with a laccase from ''E. coli''. We found papers which described the laccase [http://www.ncbi.nlm.nih.gov/protein/85674340 CueO] from ''E. coli W3110''. After blasting this laccase we found out that ''E. coli BL21(DE3)'' has this laccase, too. We decided to isolate the laccase from ''E. coli BL21(DE3)''. :* Generating new competent ''E.coli KRX cells''. :* Cultivation of ''Xanthomonas campestris B100'' and ''E. coli BL21(DE3)''. The bacterial strains we got from a working group at our University. After cultivation we isolated the genomic DNA. The DNA was needed as template for PCRs to purify the wanted laccase ORFs. :* Primer design for isolation of laccases from genomic DNA of ''Xanthomonas campestris B100'' and ''E. coli BL21(DE3)'' and for isolation of CotA from ''Bacillus pumilus ATCC7061'' from plasmid. The forward primers were designed with T7 promoter, RBS and the first 20 bases of the wanted gene after prefix. The reverse primers were designed with the last 20 bases of the wanted gene without the stop codon, a HIS-Tag, two stop codons and suffix sequence. [https://2012.igem.org/Team:Bielefeld-Germany/Protocols#Primers Primers]: Xcc_LAC_FW_T7, Xcc_LAC_RV_HIS, E.coli_LAC_FW_T7, E.coli_LAC_RV_HIS, B.pumi_LAC_FW_T7 and B. pumi_LAC_RV_HIS === Tuesday May 1th === * '''Team Student Academy:''' Searching for two plasmids with different fluorescent proteins behind and antibiotic resistance in parts registry. Found [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], a Plasmid with RFP and chloramphenicol resistance (but lacI and CAP sensitive), [http://partsregistry.org/Part:BBa_J23100 BBa_J23100], a plasmid with RFP and ampicillin resistance and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522], a Plasmid with GFP and ampicillin resistance in Kit Plate 2011. === Wednesday May 2th === * '''Team Activity Test''': Good morning everybody and welcome to the labjournal of Team Activity Tests. Today we started our work with some literature research about enzyme activity tests, laccases and its substrates. So today was filled with online research, reading papers and collecting information about the laccases our team decided to use. === Thursday May 3th === * '''Team Bacterial Laccases''': ** After the vector with the laccase gene CotA from ''Bacillus pumilus'' arrived, we transformed it into the competent ''E.coli'' KRX which we have already made competent to have a greater amount of vector. The protocol we used was as followed: *** The electroporation setup: U= 2,5kV C= 25 µF and R= 400 \omega *** Since we did not know the efficient of our competent KRX we used two different ''E.coli'' volumes for the transformation, 50µL and 100µL. We gave 50µL 10% glycerol to the reaction tubes with 1µL of the vector DNA (''Bacillus pumilus''). After the transformation we plated them on ampicillin-selection-agar-plates. ** PCR with the ''Xanthomonas campestris'' B100 and ''E. coli'' BL21(DE3) genomic DNA to isolate the laccases. Therefore we used the primers Xcc_LAC_FW_T7, Xcc_LAC_RV_HIS, E.coli_LAC_FW_T7 and E.coli_LAC_RV_HIS which are listed under Materials. ** '''PCR table''' {| class="wikitable" |- ! Material !! Volume |- | Buffer (10x Phusion) || 10µL |- | Phusion Polymerase || 0,5µL |- | dNTPs || 1µL |- | Primer Mix || 1µL |- | Template DNA || 1µL |- | DMSO || 1,5µL |- | Water || 35µL |- |} ** ''' PCR program''' {| class="wikitable" |- ! Temperature !! Time |- | 1) 98°C || 30 sec |- | 2) 98°C || 15 sec |- | 3) 62°C || 45 sec |- | 4) 72°C || 1 min |- | 5) 72°C || 3 min |- | 6) 12°C || |- |} Cycle between step 2 and 4 35 times. === Friday May 4th === '''Team Bacterial Laccases''': We did Colony PCR on the transformed the ''Bacillus pumilus'' CotA plasmid. Unfortunately the control with colony PCR didn't work. So we just picked some colonies for plasmid isolation in the hope that on the AMP plate were no false positives colonies.

Week 2

hier eine Überschrift

hier eine Zusammenfassung read more

Week 3

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 4

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 5

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 6

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 7

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 8

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 9

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 10

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 11

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 12

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 13

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 14

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 15

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 16

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 17

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 18

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 19

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 20

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 21

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 22

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 23

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 24

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 25

hier eine Überschrift

hier eine Zusammenfassung und ein read more

Week 26

hier eine Überschrift

hier eine Zusammenfassung und ein read more

55px Logo merck.jpg BioCircle.JPG Bielefeld2012 Evonik.jpg Bielefeld2012 Baxter.png Logo knauer.jpg Logo iit.jpg Bielefeld2012 BIEKUBA.jpg Logo biometra.jpg Logo bio-nrw.png Bielefeld2012 Logo ERASynbio.jpg