Testing Protein Expression Methods for Future Applications in the BASIL Biochemistry Lab Curriculum
Start Date
29-4-2022 3:45 PM
Location
Alter Hall Poster Session 2 - 3rd floor
Abstract
The Biochemistry Authentic Scientific Inquiry Lab (BASIL) curriculum consists of a series of 11 in silico and wet lab modules used to enhance the undergraduate biochemistry lab experience. These modules can be used to evaluate the function of proteins with known structures but unassign functions. Wet lab experimental procedures are used to confirm what was determined in silico, which is achieved by obtaining a workable amount of protein through expression. Two ways to induce protein expression in bacteria are by addition of IPTG after a certain cell density is reached, or by autoinduction. Autoinduction selectively increases expression of the protein of interest via lac promoters by incorporating lactose and glucose in the expression medium so there is no need to monitor the cell culture and add inducer. To test which method made more protein, red fluorescent protein was expressed under different conditions using either IPTG or autoinduction at varying initial cell densities. After growing the cells, the level of protein expression was evaluated by the size and thickness of the bands on a gel under different expression conditions. Analysis of the SDS-PAGE gel showed that autoinduction and IPTG make the same amount of protein, and the initial cell concentration does not matter. After our expression method was verified, this same method was tested with 3H04, one of the BASIL proteins. Our results indicate that autoinduction should be used for protein expression since it is quicker and easier than expression using IPTG. This would contribute to efforts of making BASIL more efficient and user friendly.
Testing Protein Expression Methods for Future Applications in the BASIL Biochemistry Lab Curriculum
Alter Hall Poster Session 2 - 3rd floor
The Biochemistry Authentic Scientific Inquiry Lab (BASIL) curriculum consists of a series of 11 in silico and wet lab modules used to enhance the undergraduate biochemistry lab experience. These modules can be used to evaluate the function of proteins with known structures but unassign functions. Wet lab experimental procedures are used to confirm what was determined in silico, which is achieved by obtaining a workable amount of protein through expression. Two ways to induce protein expression in bacteria are by addition of IPTG after a certain cell density is reached, or by autoinduction. Autoinduction selectively increases expression of the protein of interest via lac promoters by incorporating lactose and glucose in the expression medium so there is no need to monitor the cell culture and add inducer. To test which method made more protein, red fluorescent protein was expressed under different conditions using either IPTG or autoinduction at varying initial cell densities. After growing the cells, the level of protein expression was evaluated by the size and thickness of the bands on a gel under different expression conditions. Analysis of the SDS-PAGE gel showed that autoinduction and IPTG make the same amount of protein, and the initial cell concentration does not matter. After our expression method was verified, this same method was tested with 3H04, one of the BASIL proteins. Our results indicate that autoinduction should be used for protein expression since it is quicker and easier than expression using IPTG. This would contribute to efforts of making BASIL more efficient and user friendly.
