Team:Lethbridge HS/Improve
Parts Improved
We improved all of the basic parts we used by putting them into the composite parts that we used. The T7 promoter that we had at the beginning of each construct and our use of the T7 system in the Escherichia coli strain BL21(DE3) Is an improvement to each of the basic parts. This system allows us to control when the gene is being expressed. This is a great advantage to us as large batch culture overexpression is very stressful on the cells. If we did not use an inducible promoter on our genes we would see significantly less pigment than our literature would lead us to expect. This is due to the fact that the genes we are using have been optimized to produce the maximum amount of pigment possible, and this process on a single young cell would be very stressful and significantly increase the doubling time of the cells. We have induced the cells after they have replicated many times and matured, this allows us to have more cells with the construct that can produce more pigment faster.
Melanin Construct
Melanin composite part
BBa_K2481108
T7 Promoter PartBBa_I712074.
RBS Part BBa_B0034.
melA Part BBa_K193600.
Terminator Part BBa_B0015.
This construct allows us to make Melanin out of L-Tyrosine.
Figure 1. melA expression construct for use in E. coli BL21(DE3).
Anthocyanin Constructs
Anthocyanin composite part 1
BBa_K2481113
T7 Promoter Part BBa_I712074.
RBS Part BBa_B0034.
dfr Part BBa_K2481110
f3h Part BBa_K2481111
Terminator Part BBa_B0015.
Anthocyanin composite part 2
BBa_K2481114
T7 Promoter Part BBa_I712074.
RBS Part BBa_B0034.
ans Part BBa_K2481112
Terminator Part BBa_B0015.
Anthocyanin composite part 3
BBa_K2481105
T7 Promoter Part BBa_I712074.
RBS Part BBa_B0034.
3gt Part BBa_K2481002
yadH Part BBa_K2481004
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_K2481107
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_K2481106
T7 Promoter PartBBa_I712074.
RBS Part BBa_B0034.
indB Part BBa_K2481001
Terminator Part BBa_B0015.
Indigoidine composite part 2
BBa_K2481109
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. We had to split our genes into two separate composite part submissions as the size of each was too large to allow for them to be in one plasmid.
