So far during my summer of research there have a combinations of highs and lows. Leading up to now, much of my research has been spent in trying to troubleshoot the issues with my project and trying countless different approaches to attempt to successful express and purify my protein of interest. Needless to say, there were definitely some frustrating days where after a week of work and anticipation resulted in a negative result. I can proudly say I have found many ways to NOT express my protein of interest. Yet even in frustrating times there were many lessons to be learned and interesting topics to understand. Often times scientists talk about how they learn the most from their failures, and this concept has never been more evident to me.
However, not too long ago I experienced the opposite of such valleys. Having tried a new bacterial strain and additive of glucose to my cultures, I was able to express and purify my protein. It was definitely a great feeling to see positive results and to see the product of countless hours of work. This result now allows me to continue moving further into the project and approach the ultimate goal of solving a structure. With the protein expressed, I can now run different assays and experiments to further test the reactivity and nature of this particular protein.
Seeing both sides of the story in research, in failure and success, has definitely made me appreciate how much there is to learn in both regards. Failure brings knowledge and information that success can’t, and same vice versa. I look forward to continuing my project and experiencing the joys and woes of research to come.
***This is a draft, revised version emailed***
What is the structure of RAMOSA3 and what are its implications outside the trehalose pathway?
In corn, RAMOSA3 (RA3), a metabolic enzyme, is responsible for the dephosphorylation of trehalose-6-phosphate to create trehalose. Mutated versions of the RA3 protein have shown phenotypic differences in the development of corn, creating long branches at the base when compared to the wild-type corn stem. However, it is still unknown how altering this particular protein ultimately creates these phenotype differences and what exactly are the moonlighting capabilities of RA3. We suspect that RA3 has transitional regulation capabilities, whether through the regulation of RAMOSA1 (RA1), a known transcription regulator, or by its own means due to its acting upstream of RA1. We attempted many different approaches to express the plant protein RA3 into different strains of Escherichia coli (E. coli), testing many strains to find one most optimized for creating such protein. Further, we tested different conditions, altering temperature and time, in which to grow the bacteria in order to optimize protein expression. We also created a custom purification process best suited to isolate the protein of interest to run assays to test its function. Lastly, X-ray crystallography will be utilized with successfully grown crystals with RA3 in order to solve a structure. Our findings suggest that the C43 (DE3) strain of E. Coli was best suited to express this protein after being grown for three hours after induction at thirty-seven degrees Celsius. A purification process focused on the histidine tag within the protein was successful, as a process of a nickel column combined with imidazole washes isolated the protein. *Future assays and advancements in the project will allow us to reach further conclusions. At the moment, our results allow us to suspect that it will be possible to solve a structure of such protein and possibly create crystals to perform X-ray crystallography. Our future conclusions will allow us to confirm the function of RA3 as well as further develop the possibility to solving a structure.
*Assay results will be ready by poster presentation, along with possible other advancements into the project
Investigating the possibility of solving a crystal structure RAMOSA3 through analysis of expression and enzymatic activity.
RAMOSA3 (RA3) is a metabolic enzyme in maize that is responsible for the dephosphorylation of trehalose-6-phosphate to create trehalose. Mutated versions of RA3 have resulted in phenotypic differences in inflorescence branching at the base of the axillary meristem within maize. However, the transcription regulation functions of RA3 and its signaling interactions with other molecules are still unexplained, making a solved crystal structure vital for further knowledge. This study investigates the possibility of crystallization through expressing large amounts of RA3 in bacteria, purifying the protein, and confirming the function of the isolated protein. We tested expression of RA3 in multiple Escherichia coli (E. coli) through Isopropyl β-D-1-thiogalactopyranoside induction and variating temperatures and growth time. Then, we designed a purification process targeting the hexahistidine tag in RA3 to isolate the protein. Sentence about assay here. Our results show that RA3 was most optimally expressed in the C43(DE3)pLysS strain of E. Coli at 37 ℃with a 3 hour growth time and isolated with a NiNTA Agarose column followed with step elution of imidazole. Sentence about assay results here. The study suggest that crystallization is possiblewith the condition of (conclusion depends on assay results).