Team:Linkoping Sweden/Results

Firstly, we designed and ordered two different pUC57 plasmids from Genscript, one including the sequence of (EcoR1, Xba1, Promotor + RBS, His-TEV, epitope 2, Spe1 and Pst1) and the other including the sequence of (EcoR1, Xba1, Promotor + RBS, His-TEV and epitope 22, epitope 1, epitope 3, epitope 4, and epitope 17, Spe1 and Pst1). These sequences were digested by using EcoR1 and Spe1.

Furthermore, we used both Biobricks coding for red fluorescent protein which are named E1010 (RFP) and J06504 (MCherry). These were digested with Xba1 and Pst1. Finally, our linearized pSB1C3 backbone was digested with EcoR1 and Pst1 and 4 different ligations were created in the combinations (1) Promotor + RBS, His-TEV, epitope 2 and RFP, (2) Promotor + RBS, His-TEV, epitope 2 MCherry, (3) Promotor + RBS, His-TEV and epitope 22, epitope 1, epitope 3, epitope 4, and epitope 17 and RFP as well as (4) Promotor + RBS, His-TEV and epitope 22, epitope 1, epitope 3, epitope 4, and epitope 17 and MCherry.

We also decided to create another Biobrick which had no connection to our project idea but will be of great use for other iGEM participants in the future. This Biobrick consists of a His-TEV sequence and the idea is to use this Biobrick when you have a plasmid containing a protein sequence which you later on would want to express and purify, using for example a nickel-bead column. (Fig.3) This Biobrick was created by using our linearized pSB1C3 backbone and one of our ordered pUC57 plasmids. We designed primers specific only for the His-TEV sequence in our pUC57 plasmids and an overnight PCR experiment was set up for the amplification of our His-TEV sequence. The result from the PCR was controlled on an agarose-gel which indicated strongly that our PCR experiment was successful (Fig.4). Furthermore, our His-TEV sequence was digested with EcoR1 and Pst1 as well as our linearized pSB1C3 backbone and finally ligated.

All 5 of our Biobrick constructs were analyzed by transforming our plasmids into electro-competent E.Coli cells followed by a PCR colony screening. The following PCR result was controlled by running an agarose-gel which indicated plasmid weights around > 3000 Da for all the epitope and RFP/MCherry constructs as well as a weight around > 2000 Da for our His-TEV biobrick (Fig.5). Furthermore, all biobricks were analyzed by PCR screening in combination with our His-TEV sequence specific primers. This was done to prove that the ligation was successful by the appearance of a band at the weight for His-TEV on another agarose gel (Fig.6). All 5 biobricks were sent to sequencing to further prove that the cloning of our Biobricks had worked. However, unfortunately there was a problem when we were about to send our samples for sequencing. This means that we have not received any response regarding the results yet. Furthermore, since we lack information regarding the sequencing result, we have no other choice than to wait until we can begin to express proteins.