Basic Parts:

 Anthocyanin

3gt

The gene 3gt is from the anthocyanin synthesis pathway and converts the initial molecule Pelargonidin into Anthocyanin. This gene is from the organism Petunia hybrid. We have added this parts sequence to the registry for use in our composite part BBa_K1281103. The basic part is BBa_K1281003

yadH

This Gene is an Escherichia coli gene that has been shown to increase the yields of anthocyanin when paired with the genes in our anthocyanin construct. We have added the sequence to the registry for use in our composite part BBa_K1281103. The basic part is BBa_K1281002

f3h

We will be useing the gene f3h as the first gene in our anthocyanin synthesis pathway, it comes from the organism Petroselinum crispum. It was added to the registry by the 2014 Darmstadt iGEM team, part BBa_K1497009. This gene will code for a protein that converts the initial molecule flavanone into dihydroflavonol.

dfr

The gene dfr is the second one in our anthocyanin synthesis pathway. We are using the biobrick part BBa_K1497010. It was added to the registry by the 2014 Darmstadt igem team.

ans

This gene is the third gene in our pathway, it converts the molecule created by dfr into pelargonidin. It is from the organism Fragaria x ananassa and was added to the registry by the 2014 Darmstadt team. It is an engineered anthocyanidin synthase. Part BBa_K1497002.

 Zeaxanthin

crtY

This gene is from the organism Pantoea ananatis and is part or the carotenoid synthesis pathway. It is a Lycopene cyclase and converts the initial molecule Lycopene into the final molecule Beta-Carotene. This gene was added to the registry by Edinburgh 2007, part BBa_I742154.

crtZ

This gene is from the organism Pantoea ananatis and is our final gene in the carotenoid synthesis pathway. It is the beta-carotene hyroxylase, and converts Beta-carotene into Zeaxanthin. It was added to the registry by Edinburgh 2007 and is the part BBa_I742157.This part converts the Beat-Carotene into our final product, the pigment Zeaxanthin.

 Melanin

melA

The gene melA is from the organism Rhizobium etli and was added to the registry by the Cambridge 2009 iGEM team. Part BBa_K274001. This protein is a tyrosinase and converts the molecule L-tyrosine into dopaquinone, which will then polymerize into Melanin.

 Indigoidine

indB

This gene is from the organism Streptomyces chrmofuscus, and it is our original basic part submission to the registry. Its is part BBa_K1281001. It has been shown to increase the yeilds of Indigoidine when used with indC. It is a putative phosphatase.

indC

This gene is the gene that converts Glutamine into Indigoidine. It is from the organism Photohabdus luminescens and was added to the registry by the 2013 Heidelburg iGEM team. It is the indigoidine synthase and converts Glutamine thioester into the molecule %-amino-3H-pyridine-2,6-dione, two of those molecules then condense into Indigoidine. Part BBa_K1152013.

 Additional parts used

T7 Promoter

The Promoter we used for our Melanin Construct is T7 promoter, This allows us to control the production of Melanin. This promoter is from the T7 bacteriophage, it is a Virus that inserts its DNA into Bacteria in order to reproduce and stay alive. This promoter works with the T7 system inside the Escherichia coli strain BL21(DE3), (Fig 1.)


(Fig 1.) This image shows the T7 Promoter system, 1.Lactose inducible promoter 2.E. coli Ribosomal Binding site. 3.The Gene for T7 RNA polymerase. 4.Terminator 5.T7 RNA polymerase. 6.T7 promoter. 7.E. coli Ribosomal Binding site. 8.The gene/genes in our construct. 9.Terminator. 10.mRNA produced by the transcription of the construct.
This shows the process that occurs with the T7 System. We induce the Lactose inducible promoter(1) with IPTG as it mimics the lactose and cannot be broken down by the cell. This allows it to continually perform transcription on the construct(1-4) embedded in the Genome of the BL21(DE3) and thusly constantly produce the T7 RNA polymerase(5) encoded by the gene(3).This T7 RNA Polymerase(5) attaches to the T7 Promoter(6) and starts transcription on the construct(6-9) within our plasmid psB1C3. This then creates the mRNA(10) which will undergo translation and produce the protein encoded by the gene(8) due to the E. coli RBS on the construct. Part BBa_I712074.

 Ribosomal Binding Site

The RBS we have chosen for our constructs is an E. coli Ribosomal Binding Site, Part BBa_B0034

 Terminator

The terminator we have chosen for our constructs is an E. coli terminator. Part BBa_B0015, it is a double terminator made up of one BBa_B0012 terminator and a BBa_B0010 terminator.

Composite Parts:

 Anthocyanin Constructs

Anthocyanin composite part 1

BBa_K1281101
T7 Promoter Part BBa_I712074.
RBS Part BBa_B0034.
dfr Part BBa_K1497010
f3h Part BBa_K1497009
Terminator Part BBa_B0015.

Anthocyanin composite part 2

BBa_K1281102
T7 Promoter Part BBa_I712074.
RBS Part BBa_B0034.
ans Part BBa_K1497002
Terminator Part BBa_B0015.

Anthocyanin composite part 3

BBa_K1281103
T7 Promoter Part BBa_I712074.
RBS Part BBa_B0034.
3gt Part BBa_K1281003
yadH Part BBa_K1281002
Terminator Part BBa_B0015.

These composite parts come together to complete the pathway from Eriodictyol to Anthocyanin. These constructs are separated due to the size of the genes. It would be too much of a sstrain on the cell to have all of th genes in one or even two plasmids and this is why we needed three separate constructs. These constructs are each submitted

 Zeaxanthin Construct

Zeaxanthin composite part

BBa_K1281301
T7 Promoter Part BBa_I712074.
RBS Part BBa_B0034.
crtY Part BBa_I742154.
crtZ Part BBa_I742157.
Terminator Part BBa_B0015.

This construct is in the plasmid psB1C3, and will convert our initial molecule Lycopene into our final product, the pigment Zeaxanthin.

 Indigoidine Constructs

Indigoidine composite part 1

BBa_K1281201
T7 Promoter PartBBa_I712074.
RBS Part BBa_B0034.
indB Part BBa_K1281001
Terminator Part BBa_B0015.

Indigoidine composite part 2

BBa_K1281202
T7 Promoter PartBBa_I712074.
RBS Part BBa_B0034.
indC Part BBa_K1152013
Terminator Part BBa_B0015.

These two composite parts come together to convert our initial molecule Glutamine into Indigoidine. The indB has been shown to increase the yields of Indigoidine as well as it is our original basic part submissions. This construct is part BBa_K1281201 and part BBa_K1281202. We had to split our genes into tow separate composite part submissions as the size of each was too large to allow for them to be in one plasmid.

 Melanin Construct

Melanin composite part

The parts used in our Melanin construct are as follows:
T7 Promoter PartBBa_I712074.
RBS Part BBa_B0034.
MelA Part BBA_K274001.
Terminator Part BBa_B0015.

This construct allows us to make Melanin out of L-Tyrosine.