Team:IIT Madras/Results

EXPERIMENTAL RESULTS

1] Preparation of the vector (pSB1C3 + BBa_F2620)

Result 1.1: Our part of interest (BBa_F2620) was located in the 2013 Kit Plate 3, well 3P. It was reconstituted in 20 µL sterile water and 2 µL was transformed into E.coli DH5α cells. Mini-prep was performed to isolate the plasmid. The gel image is shown in Fig 1.1 below.

Fig 1.1: Plasmid Vector pSB1C3 isolation (3 kb each well)

Result 1.2: The isolated plasmid was digested with EcoR1 and Pst1 to release BBa_F2620 from the pSB1C3 backbone. Fig 1.2 (gel image) shows release of the part BBa_F2620 (1061 bp) from the plasmid pSB1C3 (2070 bp).

Fig 1.2: Restriction digestion image showing release of BBa_F2620 (2 kb+1 kb)

Result 1.3: Digestion of the plasmid vector with Spe1 and Pst1 gives the vector (3 kb) ready for ligation. Fig 1.3 shows the gel image after double digestion of the plasmid vector.

Fig 1.3: Double digested BBa_F2620 with Spe1 and Pst1 (3 kb)

Result 1.4: The vector was treated with calf intestinal alkaline phosphatase (CIP) to reduce the probability of getting self-ligated colonies. Fig 1.4 shows the final gel image of the CIP-treated vector, ready for ligation.

Fig 1.4: CIP-treated vector (3 kb)

2] Isolation of insert (Gb3 mimic, 268 bp)

Result 2.1: Gb3 mimic was delivered in pUC57 plasmid backbone. E.coli DH5α cells were transformed with pUC57. After primary culture, mini-prep was done to isolate the plasmid. Fig 2.1 shows the gel image of the isolated pUC57 plasmid containing Gb3 mimic insert.

Fig 2.1: Gel image of isolated pUC57 plasmid (3 kb)

Result 2.2: The pUC57 plasmid was then digested with Spe1 and Pst1 to show the release of Gb3 mimic insert. Fig 2.2 distinctly shows the two bands: a 2.7 kb plasmid backbone (pUC57) and a 268 bp insert (Gb3 mimic).

Fig 2.2: Digestion of pUC57 to show release of interest (268 bp)

3] Cloning of insert (Gb3 mimic, 268 bp) in vector (BBa_F2620 in pSB1C3, 3 kb) 

Result 3.1: The digested insert was eluted and a ligation reaction between the vector (3 kb) and insert (268 bp) was set up. The clones were confirmed by digesting with Spe1 and Pst1. Fig 3.1 (gel image) shows two separate bands: one at 3 kb (corresponding to vector) and another at 268 bp (corresponding to insert).
Fig 3.1: Restriction digest showing release of the cloned insert (268 bp) from vector (3 kb)

Result 3.2: The clones were further sequenced to confirm the successful cloning of the Gb3 mimic in pSB1C3. Fig 3.2 shows the sequencing results.

Fig 3.2: Sequencing of Gb3 mimic clones

4] Cloning of I3A construct (3040 bp) in pSB1C3 downstream of BBa_F2620 

Result 4.1: I3A construct (3040 bp) was delivered in pUC57. The plasmid backbone was restriction digested with EcoR1 and Pst1 to release the insert (I3A). Fig 4.1 shows the successfully isolated insert.

Fig 4.1: Gel image showing isolated I3A insert (3040 bp)

Result 4.2: The isolated insert was eluted and a ligation reaction was set up between the vector (pSB1C3+BBa_F2620, 3 kb) and the insert (I3A construct, 3040 bp). The clones were confirmed by digesting with Spe1 and Pst1. Fig 4.2 (gel image) shows two closely resolved yet separate bands. Upper band corresponds to the insert while lower band corresponds to the vector.
Fig 4.2: Restriction digest showing separate bands of vector (bottom) and insert (top)

5] Reverse-phase HPLC to detect presence of Gb3 mimic peptide 

Result 5.1: The spent medium corresponding to the induced culture was run through a C18 column. Fig 5.1 shows the chromatogram corresponding to the same. The first two peaks almost coincide with each other. The next two peaks also share a common overlap region but they are just resolved.

Fig 5.1: Chromatogram of induced culture-spent medium run through a C18 column

Result 5.2: The spent medium corresponding to the induced culture was run through a C18 column. Fig 5.1 shows the chromatogram corresponding to the same. The first two peaks almost coincide with each other. The next peak is quite small compared to the corresponding peak for the induced culture. The fourth peak, in this case, is almost negligible. It is just a tiny crest.

Fig 5.1: Chromatogram of non-induced culture-spent medium run through a C18 column.