"

From 2012.igem.org

Revision as of 07:32, 14 June 2012 (view source) Engeler (Talk | contribs) (→Week 1) ← Older edit		Revision as of 09:29, 18 June 2012 (view source) Engeler (Talk | contribs) (→Week 2) Newer edit →
Line 21:		Line 21:
	We purified our pSP-GM1 and pIYC04 plasmids from the transformed ''E. coli''.		We purified our pSP-GM1 and pIYC04 plasmids from the transformed ''E. coli''.
-	=Week 2=	+	=Week 2 11th - 15th of June =
		+	This week, the real lab work began. In order to create the biodegradable pregnancy test kit, we have three main tasks to solve. These are 1) the expression of the receptor, 2) the insertion of the indigo producing enzymes and 3) the deletion of a cell wall protein in order to enhance the chances of the ligand to pass the cell wall. Therefore we divided ourselves into three groups, the “receptor group”, the “cell wall group” and the “indigo group” each responsible for its task.
		+	Indigo group
		+
		+
		+	'''Receptor group'''
		+
		+	'''Indigo group'''
		+
		+	'''The cell wall group'''
		+
		+	We started with the construction of the knock-out fragments. First, we ran PCR with genomic yeast DNA in order to get the 500 up- and downstream fragments of the CWP2 gene. The two overlapping kanMX fragments were also amplified by PCR. For these PCR reactions, we used HPLC purified primers that were sponsored by Microsynth AG. Many thanks to Microsynth for that! After PCR the fragments were checked and purified on gel.
		+
		+	We have learned that everything takes a little bit longer than expected; it took actually the whole week to get all the four fragments, since some PCR reaction had to be repeated several times. However, we are still on schedule. On Friday, we could then start with Fusion PCR in order to fuse the fragments. Of course it did not work the first time and we have the feeling that this will be the more tricky part of the cell wall deletion part. But we will come up with some different approaches next week!
	{{Team:Chalmers-Gothenburg/footer}}		{{Team:Chalmers-Gothenburg/footer}}

---

Revision as of 09:29, 18 June 2012

Week 1

Monday, 4th of June

The laboratory work has begun and after preparing this work for several months we are all very excited to finally get it started. Today we started preparing sterile equipment and we also wrote risk declarations for all of our planned experiments. We also introduced ourselves and held a brief presentation of our project to the very nice people working at the department of System’s biology here at Chalmers University of Technology.

Tuesday, 5th of June

Today our dear yeast strains that were kindly provided to us by the Kondo research group of Kobe University, Japan, were transferred to new and fresh YPD agar plates. We also prepared buffers needed for making competent E. coli and we inoculated E. coli DH5α in LB medium.

Wednesday, 6th of June

We prepared a stock of competent E. coli DH5α. Today we also amplified the shuttle plasmids pSP-GM1 and pIYC04 that we will use in our laboratory work, by transforming E. coli DH5α. The cells were then grown on LB-Amp agar plates. We also worked with the team wiki page and created a banner and a logo for the site.

Thursday, 7th of June

Not much laboratory could be done today. Two colonies from each plate containing the E. coli DH5α that were transformed with either pSP-GM1 or pIYC04, were inoculated in ampicillin-containing media and were grown overnight. For the rest of the day, we worked with creating a team brochure and we also established a Facebook page for the team. Click here to visit our page and Like our team!

Friday, 8th of June

We purified our pSP-GM1 and pIYC04 plasmids from the transformed E. coli.

Week 2 11th - 15th of June

This week, the real lab work began. In order to create the biodegradable pregnancy test kit, we have three main tasks to solve. These are 1) the expression of the receptor, 2) the insertion of the indigo producing enzymes and 3) the deletion of a cell wall protein in order to enhance the chances of the ligand to pass the cell wall. Therefore we divided ourselves into three groups, the “receptor group”, the “cell wall group” and the “indigo group” each responsible for its task. Indigo group

Receptor group

Indigo group

The cell wall group

We started with the construction of the knock-out fragments. First, we ran PCR with genomic yeast DNA in order to get the 500 up- and downstream fragments of the CWP2 gene. The two overlapping kanMX fragments were also amplified by PCR. For these PCR reactions, we used HPLC purified primers that were sponsored by Microsynth AG. Many thanks to Microsynth for that! After PCR the fragments were checked and purified on gel.

We have learned that everything takes a little bit longer than expected; it took actually the whole week to get all the four fragments, since some PCR reaction had to be repeated several times. However, we are still on schedule. On Friday, we could then start with Fusion PCR in order to fuse the fragments. Of course it did not work the first time and we have the feeling that this will be the more tricky part of the cell wall deletion part. But we will come up with some different approaches next week!