Monday, August 12, 2019

BASIL in Ireland, Part 1

BASIL Reflection by Claire Reynolds

For my final year dissertation, I selected a challenging project which involved optimizing, purifying and characterising an unknown novel protein known as 3H04. This project presented a complex challenge which required adverse scientific knowledge and a strong understanding of scientific laboratory techniques. The project title “Novel Recombinant Protein Production, Optimization, Purification and Characterisation” appealed to me due to my keen interest in the development of novel proteins. Personally, the main objective of this project was to gain additional skills and further develop my laboratory techniques which I have gained over the past three years in my Nutraceutical course. After studying several subjects including biotechnology, biochemistry and microbiology, I have developed an in-depth understanding of the structure and function of proteins and gained insight into the expanding market of novel proteins which offer potentially new innovative treatments, functional foods and areas in the animal, plant and medical sector.
For this project to be sufficiently completed, the BASIL modules were utilised and followed to successfully achieve a final outcome. The in silico analysis tools provided by the BASIL website were primarily used at the beginning of the task to determine previous studies on the 3H04 enzyme and its predicted protein sequence, structure and function. The novel 3H04 enzyme was characterised as an alpha/ beta hydrolase by the protein data bank. This enzyme was derived from the organism Staphylococcus aureus strain Mu50 and expressed in E. coli BL21 (DE3) cells in a pMCSG19 cloning vector.

The production and optimization stages were quite tedious as optimum conditions including optimum time, temperature, optical density and Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations were essential for optimal growth. Optimization proved to be successful when recombinant protein cells were grown to an optical density of 0.4, induced with a final IPTG concentration of 1mM and shaken at 220rpm for 24 hours at 37°C. The enzyme was extremely difficult to purify and thus proved to be the most time-consuming step out of the whole project. Purification was carried out using His-Tag affinity chromatography. This step was carried out in conjunction with another final year student who was also attempting to purify a novel enzyme. After several weeks of troubleshooting the failed purification, the target protein was eluted with 50mM imidazole concentration. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to identify the molecular weight of the protein. However, this proved to be unsuccessful as only a small concentration of protein was purified. A non-reducing SDS-PAGE was then carried out and a band was visible under ultraviolet light at the 31kDa mark. This successful purification step then allowed me to move on to the final characterisation process. Throughout this process, I worked collaboratively with another final year student who was characterising his novel enzyme. Characterisation was conducted by determining the proteins optimum temperature, activity loss, solvent tolerance and pH profile. The novel protein proved to have an optimum temperature of 40°C, optimum pH 7, and could tolerate polar solvents better than non-polar solvents. A substrate specificity assay also clarified the proteins binding specificity to short chain fatty acids (p-NP acetate).

Overall, the BASIL modules collectively allowed me to successfully characterise the novel enzyme as an alpha/ beta hydrolase. I found this project challenging yet extremely rewarding when the results came together and allowed me to progress further onto each step until characterisation was finally completed. I feel the BASIL website is extremely useful and user friendly which is essential when trying to optimize, purify and characterise a novel enzyme. Now that the 3H04 enzyme has been characterised as a hydrolase, one could apply the hydrolase to a chemical reaction to determine its effects and identify if the reaction may be reversible. Having completed my dissertation, I now feel I have gained valuable experience in the biotechnology industry allowing me to pursue a career in the biotechnology, pharmaceutical industry and food sector.

Claire Reynolds completed this work as a final year project with Dr. Barry Ryan at The University of Dublin in Dublin, Ireland. Dr. Ryan became interested in BASIL after meeting Dr. Julia Koeppe from SUNY Oswego at a biophysics meeting.

Wednesday, July 10, 2019

BASIL Update from Ursinus

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Ursinus students in Rebecca Roberts's lab perform interdisciplinary research outlined in the BASIL curriculum, which is being adopted in science classrooms around the world.

Here is a link to a great article on the Ursinus College web site. I have pasted in the image and content as best I could.


Ursinus-led BASIL Curriculum Takes Root in Science Classrooms
July 10, 2019

Ursinus College is one of eight institutions that have collaborated on an inventive new biochemistry curriculum designed for lab courses at the high school, undergraduate and graduate levels.

After a number of years of development, the BASIL (Biochemistry Authentic Scientific Inquiry Lab) curriculum, developed in part by Rebecca Roberts, an associate professor of biology at Ursinus, has been published and is publicly accessible online for any institution to incorporate into their science lab classrooms.

It is an effort funded by a grant from the National Science Foundation and developed in tandem with California Polytechnic State University, San Luis Obispo; Hope College; Oral Roberts University; Purdue University; Rochester Institute of Technology; St. Mary’s University; and SUNY Oswego.

“The idea is that anyone can utilize it,” says Roberts, who is lead author on a forthcoming article about the curriculum in Biochemistry and Molecular Biology Education, a leading international journal.

In BASIL, students predict the function of a protein and then study that protein in the lab. The curriculum is flexible and can be adapted to match the available facilities, the strengths of the instructor and the learning goals of an institution, Roberts says.

At Ursinus, it’s part of two Ursinus courses: Structural Biology and Biochemistry II. The structural biology students use computational tools to investigate protein structure and deduce a possible function. Then, biochemistry students express and purify the protein and, informed by the insights of their structural biology peers, assay the protein for the proposed function.

The curriculum aims to get students to work across disciplines and transition from thinking like students to thinking like scientists, Roberts says.

In addition to the development group of institutions, the curriculum has already been adopted by one high school in Massachusetts and a college in Great Britain.

“Graduate students have also picked up on it and so have independent researchers,” Roberts says. “We’re starting to think about how to expand BASIL even more beyond the initial development team and that’s exciting.”

The BASIL initiative, she says, provides broader course-based research experiences for students.

“It’s allowing these students to have a scientific inquiry-based experience,” Roberts says. “They’re learning cutting-edge bioinformatics skills and it gets them to think cooperatively and as part of an interdisciplinary team. That’s how science works now.”

Wednesday, May 15, 2019

BASIL Starter Pack


If you are interested in trying the BASIL curriculum on your campus, you can now order a "BASIL Starter Pack" for just $25 from DNASu, thanks to our own Mike Pikaart. The kit was designed to include ten plasmids that have performed well in our undergraduate teaching labs.

Saturday, May 11, 2019

BASIL Manual


The complete BASIL student manual is now online at the following URL: https://basilbiochem.github.io/basil/, thanks to contributions from many of our members, especially to everyone who spend two very fruitful and productive writing days in Rochester in March. A special thanks to Ashley Ringer MacDonald, who build the Github site that supports the manual. Additional information is available to registered university faculty. To register, contact us from your university email address and include your name university affiliation, and the courses where you are considering using the BASIL curriculum. Please use the subject line BASIL Registration in your email.

Tuesday, April 30, 2019

News from San Antonio April 2019

BASIL was a topic of discussion at two venues in San Antonio in the same week. On Saturday April 27, Colette Daubner presented the BASIL curriculum, with rationale and results, at a symposium  titled "Enzymes, from Isotope Effects to Allostery", held in the UT Health Science San Antonio Greehey Children's Cancer Research Center. This photo shows Dr. Daubner giving her opening slide.


Then on Tuesday April 30, the end-of-year poster session was held for all students enrolled in "CURE" courses  in the Biology Department at St. Mary's University. Several teams of Biochemistry students presented their findings about unknown proteins at the session. Here are photos showing the proud teams of students:





Friday, April 26, 2019

Teach the Teachers: Python training for faculty from the Molecular Sciences Software Institute

The Molecular Sciences Software Institute (MolSSI) is offering instructor training on August 8-9, 2019 to train new instructors to teach their Python scripting and data analysis workshop.  The workshop is free and no prior Python programming experience is required!  BASIL faculty member Ashley Ringer McDonald will be one of the instructors for the workshop.  You can find out more and register here.  Registration closes July 7, 2019. 

Thursday, April 25, 2019

Active Site Alignment on the Moltimate web page

A team of four software engineering students at RIT (George Herde, Shannon McIntosh, Joshua Miller and Steven Teplica) are completing their senior capstone project with Herbert Berntstein and Paul Craig.  They have created Moltimate, a web application that performs active site alignments for query structures by comparison to a library of 942 active site templates derived from the Mechanism and Catalytic Site Atlas.

Here is a video of their final project presentation.