Team:Lethbridge/Parts

Parts Overview

In order to determine the viability of our proposed system and confirm its superiority over subcutaneous injections and current production of insulin, we designed our constructs to meet a certain set of criteria. As we hope to establish the option of using microalgae as a chassis rather than E. coli, our construct designs are used to reflect this. The goals for the fundamental design of our constructs were as follows:

• Establish a measurement system that will allow the quantification of insulin produced in a way that can be standardized.
• Ensure that our designs allow for manipulation such as purification and processing for in vitro testing and confirm proper activity.
• Develop the same constructs in both E.coli and microalgae (in this case, C. reinhardtii) to allow for comparison testing of both expression and activity.

For a complete list of these parts please see the Basicand Composite parts pages of our Wiki.

Some of our constructs were based off of parts already found in the registry such as Red Fluorescent Protein (BBa_E1010) and the proinsulin with introduced furin cut sites instead of the native cut sites (BBa_K1328003), the latter being submitted by Tsinghua 2014. Constructs include the Insulin derivatives with red fluorescent protein fusions as well as cell penetrating peptides for delivery of the insulin out of the digestive tract. All of our E.coli parts were designed with the same constitutive promoter, (BBa_J23119), RBS (BBa_J61100) and terminator (BBa_B0014) from the iGEM registry.

The microalgae constructs have the highly-expressed psaA promoter and 5’UTR (BBa_K2148000) and the rbcL 3’UTR (BBa_K2148016), which can also be found in the registry. These constructs include the same coding sequence designs as the E. coli constructs, making them comparable between both chassis.

For establishing more efficient growth of the microalgae we have also taken the NAB1 protein and placed it under constitutive expression. Constant expression of this protein decreases light harvesting antenna size which ultimately causes faster growth under high amounts of light (Mussgnug et al., 2005). An antisense construct against NAB1 was also created in order to compare growth under high light when NAB1 expression is decreased. As such, these two constructs were only designed for use in C. reinhardtii.

Table of already existing parts:

Biobrick Number	Part Name
BBa_E1010	Highly engineered mutant of red fluorescent protein from Discosoma striata (coral)
BBa_J23119	Constitutive promoter from the Anderson collection
BBa_J61100	Ribosomal Binding Site
BBa_B0014	Double terminator
BBa_K1328003	Proinsulin (modified protease recognition sites)
BBa_K2148000	psaA1 promoter + 5
BBa_K2148016	rbcL 3’UTR

For testing activity we have also designed the receptor protein that binds to insulin. As this was designed for in vitro activity assays, the construct was only designed for expression in E.coli.

More information on the constructs and our system can be found on the design page.

Our New Parts

Insulin Protein Receptor (BBa_K3237013):

The insulin receptor is a native protein found in pancreatic cells and helps regulate glucose homeostasis by interacting with insulin proteins (Ebina et al., 1985). Due to this we wanted to confirm insulin protein interactions with the receptor protein to confirm proper binding. This will ensure that furin processing allows for proper mature protein formation and the validity of our constructs for further testing.

Although there are other components to our insulin constructs such as the protein transduction domain and the red fluorescent protein, these are not present after the addition of the furin protein. For more information please see our Design page

His-mRFP1-SCI57(BBa_K3237018):

SCI57 is a stable single chain insulin variant (Hua et al., 2008) that we had decided to use for our project. As diabetes treatments can include a regime of different types of insulin being used on an individual basis we wanted to test not only the well-known proinsulin but also a single chain variant. Determining what types of insulin would be best for expression and deployment by microalgae was a key determinant of our insulin test subjects. We happily submit the SCI57 insulin for future iGEM teams in hopes that further studies in single chain but also other diabetes therapeutic strategies will be facilitated.

As we had designed each of our insulin's in E.coli and C. reinhardtii as well as with cell penetrating peptides, all parts can be considered new parts for SCI57 and are as follows in composite form:BBa_K3237018,BBa_K3237019,BBa_K3237022 and BBa_K3237023

NAB1(BBa_K3237024):

We have also looked into how we may be able to increase our growth efficiency of our microalgae production by modifying the expression of NAB1- an RNA binding protein that decreases the translation of light harvesting antenna proteins (Mussgnug et al., 2005; Beackman et al., 2009). With lower expression levels, the antenna proteins size will decrease allowing for the microalgae to tolerate brighter light. By overexpressing NAB1 in microalgae we will be able to increase production yields in environments of brighter light. We developed this to modularize the algae to optimize and control the mass of microalgae produced. This part was only designed for C. reinhardtii and has the psaA 5’UTR (BBa_K2148000) and the rbcL 3’UTR (BBa_K2148016).

NAB1 antisense (BBa_K3237012):

The NAB1 antisense construct was created in order to produce microalgae with larger light antenna's by repressing NAB1 gene expression. This control construct is transcribed into RNA which will then bind to the NAB1 gene to cause gene silencing via the dicer like gene regulation mechanism. This should allow the microalgae to grow better in lower light conditions since the antenna can be excited more easily by less photons (Mussgnug et al., 2005). The aim of this is to grow the microalgae with less energy. This part was only designed for C. reinhardtii and has the psaA 5’UTR (BBa_K2148000) and the rbcL 3’UTR (BBa_K2148016).

Our New parts associated with our designed kill switch

As we have designed a kill switch in case of accidental environmental release we have added two new genes and a new promoter sequence to the registry. We have added nucA, a DNA/RNA nuclease from Anabeaena sp. PCC 7120 (BBa_K3237029), a nuclease inhibitor from Anabeaena sp. PCC 7120 (BBa_K3237028) and a new promoter from the copMRS operon that is metal ion induced (BBa_K3237027). The nuiA gene is able to repress nucA when enough metal ions are present; under release into ponds (Čelešnik et al,. 2016), we hypothesize that the nuiA expression will be decreased or depleted and the nucA will cause cell death in the microalgae. We hope that this in tandem with the use of antisense NAB1 will prevent any propagation of insulin containing algae if accidental release were to occur.

For more information please see our design page