Title

Production and Purification of Lanthipeptide Natural Products

School Name

South Carolina Governor's School for Science and Mathematics

Grade Level

12th Grade

Presentation Topic

Cell and Molecular Biology

Presentation Type

Mentored

Abstract

Lanthipeptides are a subclass of ribosomally synthesized and post translationally modified peptides (RiPPs) that exhibit promising biological activity. Because lanthipeptides are peptides in nature, we can use protein purification methods to extract and purify them. To construct our proteins, we used a variety of distinct molecular biology techniques, including plasmid extraction, gel electrophoresis, PCR (Polymerase Chain Reaction), Gibson Assembly, Restriction Digestion, and eventually, Protein Purification. Plasmids are small, circular genetic material within bacteria that code for proteins, so we started by isolating them and performing PCR to target and amplify a specific segment. We then used a technique called Gibson Assembly to take the segment and recombine it with other gene sequences. To verify that our constructs were correct, we used a method called restriction digestion where we cut the plasmid into specific sized pieces, which could then be analyzed using gel electrophoresis. The pattern of the fragment on the gel indicated whether or not the plasmid contained the expected size insert. Then, by inserting the new strand into competent cells (cells ready to uptake genetic material), our protein could be synthesized. Our proteins contained a histidine tag, which allowed us to isolate them by using affinity chromatography. Once purified, they can be used for further in vitro discoveries.

Location

HSS 203

Start Date

4-2-2022 11:45 AM

Presentation Format

Oral Only

Group Project

Yes

COinS
 
Apr 2nd, 11:45 AM

Production and Purification of Lanthipeptide Natural Products

HSS 203

Lanthipeptides are a subclass of ribosomally synthesized and post translationally modified peptides (RiPPs) that exhibit promising biological activity. Because lanthipeptides are peptides in nature, we can use protein purification methods to extract and purify them. To construct our proteins, we used a variety of distinct molecular biology techniques, including plasmid extraction, gel electrophoresis, PCR (Polymerase Chain Reaction), Gibson Assembly, Restriction Digestion, and eventually, Protein Purification. Plasmids are small, circular genetic material within bacteria that code for proteins, so we started by isolating them and performing PCR to target and amplify a specific segment. We then used a technique called Gibson Assembly to take the segment and recombine it with other gene sequences. To verify that our constructs were correct, we used a method called restriction digestion where we cut the plasmid into specific sized pieces, which could then be analyzed using gel electrophoresis. The pattern of the fragment on the gel indicated whether or not the plasmid contained the expected size insert. Then, by inserting the new strand into competent cells (cells ready to uptake genetic material), our protein could be synthesized. Our proteins contained a histidine tag, which allowed us to isolate them by using affinity chromatography. Once purified, they can be used for further in vitro discoveries.