Team:TU Darmstadt/Notebook/LJ-Capris

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Caprolactones

April
Week 1 (2^nd of April – 8^th of April)
We found out that caprolactones are awesome so we wanted to produce them!
Week 2 (9^th of April – 15^th of April)
Searching for a paper about Caprolactone production in E. coli
Week 3 (16^th of April – 22^nd of April)
Keeping up the research. We fall more and more in love with the caprolactones.♥
Week 4 (23^rd of April – 29^th of April)
We found some interesting papers now we will have to discuss them with the rest of the groupe.

May
Week 5 (30^th of April – 6^th of Mai)
We all want to start soon, but first of all we learn some basics.
Week 6 (7^th of May – 13^th of May)
Some more research
Week 7 (14^th of May – 20^th of May)
The sequences for our two enzymes have been ordered, now we will just have to wait for them and then we can start.
Week 8 (21^st of May – 27^th of May)
Our sequences have arrived so we started working this week. First of all we diluted to ordered DNA and then we tried to clone our genes into the PSB1C3-vector. With the received vectors E. coli has been transformed.

June
Week 9 (28^th of May – 3^rd of June)
Yes! A lot of white colonies have grown. We picked them and did a colony PCR… unfortunately there was nothing to be seen. That’s why we did a PCR and redid everything we did last week. Unfortunately it did not work again…
Week 10 (4^th of June – 10^th of June)
This week we repeated everything we did the last two weeks and it didn’t work again…
Week 11 (11^th of June – 17^th of June)
We did the same as last week but we changed some condition… unfortunately we received the same results…
Week 12 (18^th of June – 24^th of June)
We thought that there might be some problems with the primers since they made a lot of homo- and hetero- dimers. That’s why we started doing the restriction digest with the sequences we orderd without multiplying them. Than we transformed E. coli with the received vectors.

July
Week 13 (25^th of June – 1^st of July)
This week we had some white colonies. We picked them and prepped them but unfortunately the prep went wrong.
Week 14 (2^nd of July – 8^th of July)
This week we repeated the prep with more experience and it did work. The positive colonies were sended in to be sequenced.
Week 15 (9^th of July – 15^th of July)
Yes! The Sequencing results are there and we successfully cloned the adh into the pSB1C3-Vector. We also made a glycerol stock from the positive ADH-Colony.
Week 16 (16^th of July – 22^nd of July)
Let’s keep up the work with our other gene chmo. This week we scanned some more colonies from the last weeks ligations via cPCR and Control Direst… unfortunately there was no positive result.
Week 17 (23^rd of July – 29^th of July)
This week we gave up on our old colonies and did a restriction and ligation of chmo into the pSB1C3 backbone again. When analyzing it we did not see anything on the agarose gel.

August
Week 18 (30^th of July – 5^th of August)
Don’t give up on chmo, let’s try cloning it again. After transforming E. coli with it we did two cPCRs with different conditions… both failed.
Week 19 (6^th of August – 12^th of August)
Adh has been clones into a pSB1A3 vector two allow us to clone both genes on one backbone. Futhermore adh has been transformed into BL21 for later expression. We also decided to order chmo with Gibson assembly overhangs to have an alternative way to clone it into pSB1C3.
Week 20 (13^th of August – 19^th of August)
This week our new sequences of chmo arrived and we tried gibson assembly. The cPCR went bad. A SDS-PAGE has been done with ADH.
Week 21 (20^th of August – 26^th of August)
Some of the colonies received with the Gibson assembly seemed positive so we send them in to be sequenced.

September
Week 22 (27^th of August – 2^nd of September)
With the beginning of this month something exiting happened: we received a vector form the working group of Bornscheuer from the University of Greifswald, which has adh as well as chmo cloned on it so we could start our assays. These we transformed into BL21 and did a glycerol stock with them.
Week 23 (3^rd of September – 9^th of September)
We did a SDS-Page with our ADH-colonies, which failed. Then we did a SDS-PAGE with the received vector which failed as well.
Week 25 (10^th of September – 16^th of September)
We redid the SDS-PAGE with adh and chmo. Afterwards we did a westernblot which failed.
Week 26 (17^th of September – 23^rd of September)
Let’s retry cloning chmo into pSB1C3. We also did a SDS-PAGE again. The following westernblot failed.
Week 27 (24^th of September – 30^th of September)
Since the last weeks cloning went wrong we redid that and finally were able to prove via cPCR and control digest some colonies which had chmo in them. These were sended to be sequenced. Another success this week was our westernblot with our received plasmid.

October
Week 28 (1^st of October – 7^th of October)
Yes great success! The chmo sequencing has been successful. At the beginning of this week we also did a cleanup of our enzymes. And during the week we run some assays which were not promising yet. And since it is almost the end of the iGEM year we further more did a westernblot of adh and chmo of our own vactors, unfortunately there was nothing to be seen
Week 29 (8^th of October – 14^th of October)
Last week in the lab! We finally received positive results from the assays. We also redid the western blot and try to save the one from last week.
Week 30 (15^th of October – 21^st of October)
… and cleaning of the Lab
Week 31 (22^nd of October – 28^th of October)
Fly to Boston and attend the Giant Jamboree!