Team:Baltimore BioCrew/Improve

2020 Baltimore Biocrew

Improving A Part

Improving A Part

Goal/Motivation

Our newly designed phytoplankton will grow in iron-poor regions by expressing genes that code for iron-uptaking proteins. The concern our team addresses is that the creation and implementation of such high-performing phytoplankton into the ocean could eventually prove to be problematic. Without a switch to keep the growth of the new phytoplankton in check, an algal bloom could occur, eventually decreasing the amount of oxygen in the aquatic ecosystem. In order to avoid this, we are developing a method of sensing and controlling phytoplankton’s use of iron.

Our subteam is improving upon an iron-sensing promoter system to make it more sensitive to several increasing iron levels. We could use this promoter system upstream of the genes we’re expressing to promote phytoplankton growth -- so growth is only stimulated if iron concentrations are below a specific threshold. This promoter system would also have major applications, including monitoring iron nutrient levels and predicting future algal blooms. To do this, we need a way of sensing how much iron a cell is taking in and using.

Background

To sense iron levels, we are improving upon promoters that have binding site(s) for Ferric Uptake Regulator (FUR) protein. Ferric Uptake Regulators are a large family of proteins that can be activated by metals to bind DNA, controlling the expression of a diverse set of genes (Lucarelli et al., 2008). FUR can bind with iron (Fe2+) which then binds to its binding site on DNA (the “FUR box”) (Figure 1). Since the FUR box is typically within the promoter region of genes, FUR and Fe+2 binding the FUR box results in repressing the expression of the downstream gene(s) (Figure 2).

Our subteam is improving upon an iron-sensing promoter system to make it more sensitive to several increasing iron levels. We could use this promoter system upstream of the genes we’re expressing to promote phytoplankton growth -- so growth is only stimulated if iron concentrations are below a specific threshold. This promoter system would also have major applications, including monitoring iron nutrient levels and predicting future algal blooms. To do this, we need a way of sensing how much iron a cell is taking in and using.

FUR Binding Site
Figure 1: Repeated FUR binding sites in DNA. Source: 2013 Evry team

Previous teams have developed iron-sensitive promoters that use FUR binding sites. The 2013 Evry iGEM team used the AceB promoter upstream of GFP (BBa_K1163102). Although this team was able to show increasing iron levels decreased GFP expression using their promoter, it was unclear where FUR bound in their sequence (contained no identifiable Fur box), and therefore difficult to improve upon. We focused instead on the 2018 ECUST’s iGEM team, which inserted FUR boxes into a constitutive promoter, creating a series of iron-sensitive promoters (pfur1 - BBa_K2737003, pfur2 - BBa_K2737004, pfur3 - BBa_K2737005). These promoters were shown to generate a modest decrease in fluorescence when under the influence of a very high iron concentration (10 µM). We are interested in seeing if we can make these promoters more sensitive to lower iron concentrations.

Inhibition of the pfur Promoter
Figure 2: Diagram showing the inhibition of the pfur promoter. (Left) With the absence of Fe(II), FUR cannot bind to the FUR Box, and gene expression is uninhibited. (Right) The Fe(II) molecule binds to the FUR ligand, which binds to the FUR Box, inhibiting the promoter system.

Design of Our Promoters

We are improving upon the pfur promoter constructs created by the ECUST 2018 team. This team created three promoters (pfur1 - BBa_K2737003, pfur2 - BBa_K2737004, pfur3 - BBa_K2737005) that varied in where the FUR box was located within the promoter region. We aim to make these promoters more sensitive to iron levels by varying the number and location of iron binding sites (Figure 3).

Improvements to previous iron-sensitive promoters
Figure 3: Our improvements to previous iron-sensitive promoters. (a) Promoters created by ECUST 2018 team, where the -35 and -10 regions represent the sequences “atggcg” and “tggcatgat,” respectively. (b-c) The promoter we are creating and characterizing.

Testing Our Promoter Performance

To test our promoters, we synthesized constructs where each promoter was put upstream of a red fluorescent protein (mCherry - BBa_K3651025) (Figure 4)

Iron-sensitive test constructs
Figure 4: Iron-sensitive test constructs. We created 7 composite parts by putting our iron-sensitive promoters (including ECUST 2018’s) upstream of a red fluorescent protein (mCherry).

This yielded the following “Test Constructs” that we are in the process of verifying using gel electrophoresis. We also plan to include and test a negative and positive control, as described in the table below.

  • Table 1: Test Constructs
    Device Function Bio Brick # Short description
    Negative Control BBa_R0040 "TetR repressible promoter"
    Positive Control BBa_K3651036 J23100_mcherry
    Test Construct 1 BBa_K3651026 pfur1-mCherry
    Test Construct 2 BBa_K3651028 pfur2-mCherry
    Test Construct 3 BBa_K3651037 pfur13-mCherry
    Test Construct 4 BBa_K3651035 pfur23-mCherry
    Test Construct 5 Bba_K3651029 pfur12-mCherry
    Test Construct 6 Bba_K3651031 pfur123-mCherry
    Test Construct 7 BBa_K3651038 pfur3-mCherry

For this experiment, we are comparing how much red fluorescence is produced by bacteria transformed with each construct and grown in varying iron concentrations. We picked colonies of transformed bacteria and grew them in liquid cultures with four iron concentrations: no iron (0 µM), low iron (0.1 µM), medium iron (1 µM), and high iron (10 µM). We grew these cultures overnight (for 16 hours, at 37C, 220 rpm). You can view our full protocol here.

We obtained positive colonies for each of our genes (see image below). Two samples were taken from each plate and amplified via PCR, and as you can see, the results were strong.