Team:Baltimore BioCrew/Parts

2020 Baltimore Biocrew

parts header

Parts

New Parts

isiB (Flavodoxin)
Flavodoxin is a low-potential electron donor for redox enzymes. It can substitute for ferrodoxin in times of low iron, however it is not thought to be "essential" to organism survival even in these times. Composite part: BBa_K3651030

ZupT
It is known to be a Fe+2 transporter gene along with feoA and feoB, and is suggested to facilitate zinc uptake. It may also transport other divalent cations such as the ions of copper and cadmium and is said to be involved with the activity of copper ion and ferrous iron transmembrane transport (Ahlgren et al., 2019). Composite part: BBa_K3651011

idiA
It codes for the Iron uptake protein A2, which belongs to the bacterial solute-binding protein 1 family. It plays an important role in protecting the acceptor side of photosystem II against oxidative damage, especially under iron-limiting growth conditions, and may also play a role in copper supply to thylakoid proteins. It has been documented that transcription of this gene increases when cells are grown in the absence of iron. Composite part: BBa_K3651012

feoA
It is known to be a transport gene for transition metal ion binding, along with feoB. FeoA functions as a transition metal ion binding gene, and it may be of significance in the reductive uptake pathway, where Fe(III) is reduced and then transported as Fe(II). Composite part: BBa_K3651032

feoB
It a large membrane protein that is capable of hydrolysing GTP, and one of three parts of the Ferrous Iron Transport system. It is useful to bacteria living in anaerobic or low pH conditions where the Fe2+ form of iron is dominant, and works along with feoA and feoC. Composite part: BBa_K3651014

tonB
It is known to be part of an energy-converting complex that allows for the active transport of iron siderophores and other nutrients (Noinaj et al., 2010). All of the tonB complexes are regulated by Fur and are repressed when the iron amount reaches a certain level. Composite part: BBa_K3651015

Ferritin
It is an iron-storage protein whose ferroxidase center binds Fe2+ ions, oxidizes them by dioxygen to Fe3+, and participates in the subsequent Fe3+ oxide mineral core formation within the central cavity of the protein complex. It also catalyses the Fe2+ oxidation reaction. Composite part: BBa_K3651033




Improved Parts


Basic Parts

Ferric Uptake Regulator (FUR) is a protein that binds with Fe2+ which then binds to the FUR box. Since the FUR box is typically within a promoter region of genes, FUR and Fe+2 binding the FUR box results in repressing the expression of the following gene.

These DNA sequences were created by emulating the design similar genes from the ECUST 2018 team.

Pfur12
For Pfur12, the first FUR box is between what ECUST 2018 calls the-35 and -10 region and the second FUR box downstream of the -10 region. If this promoter is placed upstream of LacI, then the expression of LacI is repressed by the presence of the FUR protein and ferrous iron in the cytoplasm.

Pfur13
For Pfur13, the first fur box upstream of what ECUST 2018 calls the -35 region and the second fur box between the -35 and -10 regions. If this promoter is placed upstream of LacI, then the expression of LacI can be regulated by the presence of the Fur protein and ferrous iron in the cytoplasm.

Pfur23
For Pfur23, the first fur box upstream of what ECUST 2018 calls the -35 region and the second fur box downstream of the -10 region. If this promoter is placed upstream of LacI, then the expression of LacI can be regulated by the presence of the Fur protein and ferrous iron in the cytoplasm.

Pfur123
For Pfur123, the first fur box upstream of what ECUST 2018 calls the-35 region, the second fur box between the -35 and -10 region, and the third fur box downstream of the -10 region. If this promoter is placed upstream of LacI, then the expression of LacI can be regulated by the presence of the Fur protein and ferrous iron in the cytoplasm.

mCherry Optimized
This is a red fluorescence protein that has been codon optimized for expression in E.Coli. This can be used to express red fluorescence if put downstream of a promoter, ribosome binding site, etc.. In our project, this was used as a marker for promoter activity.


Composite Parts

These transcription unit include an iron sensitive promoter with one Ferric Uptake Regulator (FUR) binding site upstream of a ribosome binding site (BBa_J61100), red fluorescence protein (BBa_K3651025), and terminator (BBa_B0010). Altogether, this is an iron sensor. When iron concentration increases (in the presence of FUR protein), red fluorescence decreases. The FUR Protein in presence of iron will block the transcription because it will bind to its FUR Box.

Pfur1-mCherry


Pfur2-mCherry


Pfur3-mCherry


Pfur12-mCherry


Pfur13-mCherry


Pfur23-mCherry


Pfur123-mCherry


J23100-FUR
This is a part that constitutively expresses Ferric Uptake Regulator Protein (FUR). In the presence of iron, iron-bound FUR can bind to a FUR box and block transcription. These FUR boxes are typically within promoters. We could use this part in conjunction with pfur1-mcherry (BBa_K3651026) to confer iron-sensitive expression of mcherry.

J23100_mCherry
This part constitutively expresses mcherry. This part includes a constitutive promoter that is part of the Anderson family (BBa_J23100) upstream of a codon-optimized mcherry (BBa_K3651025) . In our project, we used this part as a positive control so that we could see mcherry being maximally expressed.