Performing Department
(N/A)
Non Technical Summary
Silk has been an important material throughout history, used in textiles, cosmetics, and medical applications. Currently, the global silk market is expanding as groups aim to find a moreenvironmentally friendly alternativeto synthetic materials.However, the United States has faced challenges in developing a successful silk manufacturing industry, missing out on the expanding global market. Highlightingthe need for innovative approaches to silk production.Traditionally, silk manufacturing relies on the domesticated silkworm, Bombyx mori. Current silk farming practices are labor-intensive and not well-suited for high labor cost areas like the USA. Additionally, outdoor silk farming faces challenges such as inconsistent quality and supply chain issues, which can be exaserbated by climate change. Furthermore, recent advancements in genetic engineering, like CRISPR technologies, have opened new possibilities for using silkworms to produce valuable proteins. However, producing large proteins with common methods, such as using E. coli bacteria, is difficult. Silk glands, on the other hand, are well-suited for this task.In my project, I aim to leverage The Indian meal moth, Plodia interpunctella (Pi), as an alternative silk producer for both nutural and modified silk. Pi can be easily raised indoors with controlled conditions and standardized food sources. It is also a good model for genetic engineering, supported by extensive genetic data. By understanding how to enhance silk production and developing methods for collecting silk and expressing modified silk proteins, Pi could help the USA enter the global silk market. This approach would offer better standardization and industrial manufacturing processes while minimizing the release of genetically modified organisms into the environment.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
15%
Developmental
15%
Goals / Objectives
Silk is a historically important material that has been utilized throughout human history for a variety of applications including in the textile, cosmetics, and biomedical industries and offers a greener alternative to synthetic polymers. Because of this, improving silk production and manufacturing has been a global goal and challenge for thousands of years. However, introduction of silk manufacturing into the United States of America (USA) has been wrought with challenges, leaving the USA out of the large and currently expanding global market and underscores the need for innovative approaches for silk production.Despite the expansive biodiversity found in silk producing organisms, industrial manufacturing primarily relies on the domesticated silkworm,Bombyx mori, where its current sericulture practices remain labor-intensive and largely incompatible with regions where labor costs are high, such as the USA. Moreover, the reliance on outdoor sericulture practices introduces quality standardization and supply chain challenges, exacerbated by shifting environmental factors driven by climate change. Additionally, with recent advancements in genome editing technologies, particularly CRISPR/Cas, leveraging silkworms as bioreactors to produce valuable recombinant proteins has been opened. One major challenge with recombinant protein expression in most common host vectors, particularlyE. coli, is the inability to produce large molecular weight proteins.This is a space where silk glands have evolved to thrive and can be leveraged to produce challenging proteins.MajorGoal:Leverage the alternative silk producing species Plodia interpunctella(Pi) as a novel silk producer and bioreactor for recombinant silk-fiibroin like proteins.With its well-established indoor laboratory rearing protocols,offering controlled environmental conditions and standardized food sources, as well as its utility as a genetic engineering model organism,supported by robust genomic sequencing data. Understanding how to promote silk production, collection protocols, and limitations with genetically modified silk protein expression will allowPito be an innovative solution for the USA to expand into the silk global market with better standardization and industrial manufacturing protocols that limit release of genetically modified organisms. Objectives:EvaluatePlodia interpunctellasilk production and ease of collection temporally, as a function of rearing chamber surface area. Evaluation will be characterized by total silk mass, fiber diameter and density, and silk fibroin protein gene expression.Development of 3D printed inserts: Within our standard rearing chamberswe aim to increase surface area within the chambers by the introduction of 3D printed PLA (polylactic acid) inserts to determine whether increasing surface area positively correlates with the increase of silk, while holding other optimized conditions constant. Design of inserts will be conducted and produced using the core facilities offered at the University of Florida where 4 different designs will be created.Evaluate silk production in boxes with the various 3D printed inserts.Compare wild-type silk production to genetically modified silk proteins inPlodia interpunctella, with a focus on how the length of expressed protein influences total silk output.?Identification of Fibroin heavy PAM sequences: Utilizing the Kawahara et al.2023, PAM sequences and subsequent sgRNA's will be designed to target the middle region and 3' prime region of theFib-Hgene.Insertion to modify Fib-H length:Kawahara et al.36annotated the Fib-H gene forPiand determined the native size was ~420kDa. We aim to utilize the HDR CRISPR/Cas9 pathways to modulate this length from ~100 kDa to ~ 600 kDa. At the sites upstream of the native stop codon, we will provide DNA template that will generate a stop sequence to truncate the protein. To increase the size from the native structure we will introduce the native repeating sequence found inPiuntil we reach the desired length and then end the protein with a stop sequence.?
Project Methods
Efforts: Objective 1:As we look towards utilizingP. interpunctellato produce silk on an industrial scale, further optimization needs to be accomplished.AsP.interpunctellaproduces silk as a function of wandering around their habitat, I aim to understand whether surface area within their habitat will influence the total amount of silk they will produce.To accomplish this, I will develop3D printed inserts: Within our standard rearing chambers we aim to increase surface area within the chambers by the introduction of 3D printed PLA (polylactic acid) inserts to determine whether increasing surface area positively correlates with the increase of silk, while holding other optimized conditions constant. Design of inserts will be conducted and produced using the core facilities offered at the University of Florida where 4 different designs will be created.The inserts will be added to the rearing containers and our P. interpunctella colony will be reared as consistently as described in our previous publication. Once the larvae have matured to the pupal stage and no longer are depositing wandering silk, we will collect the silk to get a mass of how much silk is being produced as a function of surface area.Efforts: Objective 2:As we examinePias a potential bioreactor to produce recombinant protein, a critical starting point is to examine how changing the length of theFibroin heavygene and therefore the molecular weight will influence silk expression and production.Identification of Fibroin heavy PAM sequences: Utilizing the Kawahara et al.2023, PAM sequences and subsequent sgRNA's will be designed to target the middle region and 3' prime region of theFib-Hgene.Insertion to modify Fib-H length:Kawahara et al.annotated the Fib-H gene forPiand determined the native size was ~420kDa. We aim to utilize the HDR CRISPR/Cas9 pathways to modulate this length from ~100 kDa to ~ 600 kDa. At the sites upstream of the native stop codon, we will provide DNA template that will generate a stop sequence to truncate the protein. To increase the size from the native structure we will introduce the native repeating sequence found inPiuntil we reach the desired length and then end the protein with a stop sequence.Within this aim we will characterize the integration of new sequences through sequencing and protein analysis such as SDS-PAGE. Additionally, we will conduct an assessment of silk production compared to the maximum silk group that is reported in aim 1.All data in this work will be graphed using GraphPad Prism and statistically signifiance of the data will be assigned using this software.