Team:IISER Bhopal/Results

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Results

Trigger Factor of Pseudoalteromonas haloplanktis

Immediately after synthesizing the TF we had to transform it into the appropriate cells. Once transformed, plasmid extraction was performed and the gel obtained may be seen in Figure 1.1. Post extraction, the plasmid was employed to transform cells, and its effect on cell growth was observed throughout the log phase. Our literature survey and IHP had helped us understand that optimal yield of different proteins is subject to different growth conditions - hence it was necessary for us to perform growth curve analysis and observe the effect of the Trigger Factor's induction on cell growth. The observations obtained for the same are given below. The temperatures chosen were 37°C (as a model for optimal conditions) and 18°C (as a model for suboptimal conditions). While we ideally would have liked to achieve even lower temperatures (as our model clearly predicts that TF could survive near 0°C-like conditions), we were most unfortunately limited by the available infrastructural facilities.

Fig.1.1: Gel image of Trigger Factor P.haloplanktis cloned in pUC18.

Fig.1.2: Trigger Factor P.haloplanktis cloned in pUC18.

Growth Curve at 37°C

Growth Curve at 18°C

Cpn60

Cpn 60 was successfully digested from the parent backbone pSB1C3, which was confirmed on gel via size confirmation and sequencing exercises. As this part had already been submitted by the 2011 Amsterdam team, we decided to further characterize it (as a part of the Bronze medal criteria) and check how its induction affected cell growth. The results of the growth analysis may be seen in the graphs provided below. The temperatures chosen were 37°C (as a model for optimal conditions) and 18°C (as a model for suboptimal conditions). While we ideally would have liked to achieve even lower temperatures (as our model clearly predicts that TF could survive near 0°C-like conditions), we were most unfortunately limited by the available infrastructural facilities.

Fig.2.1: Gel Image of Cpn60 cloned in pUC18

Fig.2.2: Cpn60 cloned in pUC18

Growth Curve at 37°C

Growth Curve at 18°C

Cpn10/Cpn60

We obtained the Cpn 10 /60 by sequential cloning of Cpn 60 and Cpn 10 into pUC18 plasmid (in that particular order). Similar to GroEL/GroES, they function by associating with each other so as to enable better folding of proteins. We characterized the composite part and checked how its induction affected cell growth. The results of the growth analysis may be seen in the graphs provided below. The temperatures chosen were 37°C (as a model for optimal conditions) and 18°C (as a model for suboptimal conditions). While we ideally would have liked to achieve even lower temperatures (as our model clearly predicts that TF could survive near 0°C-like conditions), we were most unfortunately limited by the available infrastructural facilities.

Fig.3.1: Gel Image of Cpn10/60 cloned in pUC18

Fig.3.2: Cpn10/60 cloned in pUC18

Growth Curve at 37°C

Growth Curve at 18°C

Chaperonin Comparitive Analysis

For a wholesome understanding of how different chaperones peak at different temperatures, we set up a 96 well experiment to check the same. A growth curve was plotted for three different temperatures, this data was also used by our modelling team to extrapolate the minimum temperature at which the cells could survive. An SDS page was run for the whole cell lysates of the samples to confirm the presence of their respective proteins.

Growth Curve at 37°C

Growth Curve at 23°C

Growth Curve at 18°C

Fig.4.1: Results for SDS-PAGE of whole cell lysates of cell lines containing TF, Cpn60 and empty vector(pUC18) with Rosetta as a control.

@iGEM IISERB