Team:Linkoping Sweden/Results

Our vision is to create a biobrick including the sequence of the Ara h1 protein linked to a red fluorescent protein so that we in turn can express the protein and thus provide an interaction of this protein complex with the antibodies. To ensure ourselves that this epitope-red fluorescent protein complex will bind to the Ara h1 specific IgG antibodies several ideas of biobrick-setup was brought to mind. Since we use both monoclonal antibodies specific for epitope 2 of Ara h1 and polyclonal antibodies specific for several epitopes of Ara h1 we decided to create a biobrick consisting of epitope 2 of Ara h1 linked to a red fluorescent protein as well as a biobrick consisting of five wisely chosen epitopes (epitope 22, epitope 1, epitope 3, epitope 4, and epitope 17) of Ara h1 linked to a red fluorescent protein. However, since there are two different mutants of the red fluorescent protein (called RFP and MCherry) we decided to create two setups of every biobrick combination to ensure that the best possible detection by FRET is used since RFP and MCherry slightly differs in their wavelength areas. The main idea is to practically test this FRET effect in both epitope-RFP and epitope-MCherry combinations by fluorescence which hopefully will prove to us which mutant is best suited to use and thus prove that our theory actually works.

'''Biobricks''' These discussed combinations will result in 4 different biobricks which are the two combinations of RFP linked to either epitope 2(Fig.1) or all 5 epitopes (Fig.2) as well as the two combinations of MCherry with either epitope 2(Fig.1) or all 5 epitopes (Fig2). The protein expressed from the biobrick including epitope 2 linked to RFP/MCherry will thus be used in combination with the monoclonal antibodies which are specific for epitope 2 as mentioned previously and the 5 different epitopes linked to RFP/MCherry will be used in combination with the polyclonal antibodies. Worth mentioning as well is that all the 4 biobricks will contain a sequence for Promotor + RBS as well as a sequence for His-TEV. (Bild på sekvenserna)

'''Approach''' First of all 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 the use of EcoR1 and Spe1. Furthermore, we used both biobricks of the red fluorescent protein named E1010 (RFP) and J06504 (MCherry) which 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. Furthermore, we decided to create another biobrick as well 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 by the use of for example a nickel-bead column. (Fig.3) This biobrick was created by the use of 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 as well to enable ourselves to prove that the ligation was successful by the appearance of a band at the weight for His-TEV on another agarosegel (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 on sequencing which 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. (Bild på gelerna)

'''Antibody labeling''' Our project idea is also based on the labeling of our antibodies with a green fluorescent probe called FITC. The labeling was performed by following the labeling protocol from Thermo Scientific and the result was analyzed by measuring the absorbance. In (Fig.7) one can see the result from the FITC labeled polyclonal antibodies in which the concentration of FITC at Amax (494 nm) was calculated to be 11.79 microM as well as the calculated protein concentration at A280 (277.5) turned out to be 5.6 microM, indicating a 2:1 ratio of labeling. In Fig. 5 one can see the result from the FITC labeled monoclonal antibodies in which the concentration of FITC at Amax (493 nm) was calculated to be 5.0 microM as well as the calculated protein concentration at A280 (278.5) turned out to be 4.7 microM, indicating a 1:1 ratio of labeling. Our goal is to test our FRET effect by expressing our RFP/MCherry linked epitope protein before entering the competition in Boston. Hopefully we will be able to perform this before the deadline, otherwise we will continue with our project afterwards. (Bild på rör med inmärkta antikroppar och abs mätning) .