PROS by the Stony Brook University 2022 iGEM Team

Contributions

Over the course of the past year, our team has made considerable contributions to future iGEM teams. We have introduced different parts to the registry, increased understanding of new cell lines for future teams, including collecting high quality protocols, developed various mathematical modeling guides, and documented and added (to the iGEM registry) the baculovirus expression system for recombinant human proteins. Overall, this page contains the list of all registered parts from the Stony_Brook Team, description of the used cell lines and mathematical models, and lists of protocols available.

Parts

SF9 Parts

Basic Parts

Part Name Registry ID Type
New Parts
Human Protein S Gene (PROS1) BBa_K4235000 Coding
Polyhedrin Promoter BBa_K4235001 Regulatory
Gentamicin Resistance Gene BBa_K4235003 Coding
SV40 BBa_K4235020 Terminator
Tn7R BBa_K4235026 DNA (terminal inverted transposon elements)
Tn7L BBa_K4235027 DNA (terminal inverted transposon elements)
LIC Primer (insert 5’) BBa_K4235031 Primer
LIC Primer (insert 3’) BBa_K4235032 Primer
LIC Primer (vector 3’) BBa_K4235033 Primer
LIC Primer (vector 5’) BBa_K4235034 Primer
Contribution to Existing Parts
pC Promoter BBa_K3814029 Regulatory
AmpR Promoter BBa_K2308010 Regulatory
Ampicillin Resistance Gene BBa_I722014 Coding
Kozak Sequence BBa_K4046050 RBS

Composite Parts

Part Name Registry ID Type
New Parts
Gentamicin Resistance Gene Cassette BBa_K4235024 Coding
Protein S Gene Expression Cassette BBa_K4235007 Coding
Ampicillin Resistance Gene Cassette BBa_K4235025 Coding
Mini-attTn7 Segment BBa_K4235010 Coding
pFastBac-Htb_miniTn7 BBa_K4235002 Plasmid

E. coli Parts

Basic Parts

Part Name Registry ID Type
New Parts
Human Protein S Gene (PROS1) BBa_K4235011 Coding
AmpR Promoter BBa_K4235017 Regulatory
Ampicillin Resistance Gene BBa_K4235018 Coding
LIC Insert Reverse Primer ( 3’) BBa_K4235036 Primer
LIC Insert Forward Primer (5') BBa_K4235035 Primer
Contribution to Existing Parts
T7 Promoter BBa_J64997 Regulatory
T7 Terminator BBa_B0016 Terminator
RBS BBa_K2927001 RBS

Composite Parts

Part Name Registry ID Type
New Parts
PROS1 Expression Cassette BBa_K4235030 Coding
Ampicillin Resistance Gene Cassette BBa_K4235029 Coding
pET His6 LIC Cloning Vector (2Bc-T) BBa_K4235016 Plamid

Plasmid - Bac to Bac SF9 Expression System

We received the baculovirus transfer vector pFastBac YMBac-II as a kind donation from the Airola lab at Stony Brook University. pFastBac is used for the expression of 6x His-TEV-tagged proteins in insect cells through the Bac-to-Bac baculovirus expression system.

Organisms and Expression Systems

Origami Cells, Strain B2

We set the precedent for other iGEM teams to use the origami E. coli strain B2, if the protein being expressed has multiple disulfide bonds and/or complex folding. We tried expressing protein S (a complex, secreted, monomeric human protein with multiple disulfide bonds) in BL21 and in the origami strain. Use of the BL21 strain was unsuccessful. We only achieved results when using the origami strain. This could be due to BL21’s inability to fold the protein properly and inability to create disulfide bonds, which may have contributed to excessive aggregation, therefore increasing toxicity of this protein for the BL21 E. coli. Future teams that are trying to express proteins with multiple disulfide bonds in E. Coli may want to consider using origami cells over BL21.

SF9 Insect Cells

Stony_Brook is one of the few iGEM teams to have added SF9 parts to the registry. We have studied SF9 use for protein expression extensively and acquired significant resources, such as the necessary protocols, for use of this system. We have set a precedent for all future teams that wish to express proteins that require glycosylation, as prokaryotic cells cannot provide folding and complex post-translational modifications. Using prokaryotes is beneficial for lowering the costs of protein production. This was beneficial for our specific project, as our focus is to industrialize the process. However, at times, because of the protein’s potential complexity, it may be more reasonable to use SF9 cells to produce such complex proteins.

Baculovirus Expression System (Bac to Bac)

After a lot of research and consulting with advisors and experts in the field, we decided to use the baculovirus expression system for the SF9 insect cell line (Spodoptera Frugiperda) and E. coli strains, BL21 and origami B2. SF9 being a eukaryotic cell line, is popular for the expression of human proteins and antibodies because of its ability to perform advanced post-translational modifications, which prokaryotic hosts like E. coli are incapable of. However, cloning and expression using the baculovirus systems in SF9 cells includes multiple additional extra steps than the traditional bacterial expression systems and generates time constraints for successful completion of the project. Furthermore, the use of SF9 cells is not as easy to scale up industrially and prokaryotes such as E. coli are a preferred organism for up-scaled industrial production, which is the ultimate goal of our project. Therefore, we decided to express protein S in E. coli Bl21 and origami B cell lines along with SF9 cells, which would provide us with comparative data between the efficiency and feasibility of protein production in eukaryotic vs. prokaryotic hosts.

Recombinant DNA technology has become widely used in modifying baculoviruses for heterologous gene expression in insect cells. Baculovirus expression systems have multiple benefits over using traditional bacterial expression systems and other eukaryotic expression systems:

  • Baculoviruses have a restricted host infection range, therefore, they are relatively safe to work with since they do not infect vertebrates.
  • Baculovirus protein expression is driven by a very potent and strong polyhedrin promoter, allowing for high levels of expression compared to other expression systems.
  • Baculovirus genome expresses heterologous genes while diminishing host protein expression. This makes protein recovery from infected cells easier.
  • Moreover, insect cells can be infected with baculoviruses containing two or more gene expression circuits or simultaneously infected with multiple viral genomes containing different gene expression circuits. This allows for expression of multiple proteins in a single infection cycle.
  • Baculoviruses are not limited by the size of the protein they can functionally express. Baculoviruses infection of insect cells can achieve recombinant proteins with accurate post-translational modifications, resembling proteins secreted in higher eukaryotes, including humans.

Protocols

Here we list the protocols which we gathered over the course of our project. We would like to give special thanks to our advisors and friendly scientists who offered their protocols and/or knowledge to build protocols.

E. coli

SF9

Mathematical Modeling Guides

We have created guides for mathematical modeling of protein expression. These have been used by multiple iGEM teams which have collaborated with us. The guides outline the biological processes to be modeled, the background knowledge needed to understand how to model them (both biological and mathematical), and how to model them. They also include guidance on what assumptions need to be made, and what constants need to be specified for use in the model, etc. Below is an image from one of our guides that summarizes the covered content. Our guides can also be found at the following links:

Discover

Team

The members of the 2022 Stony Brook University iGEM Team

Description

Introduction to our project.

Access Wiki Tools View Source Code