Team:Lethbridge HS/Improve

Improved Parts

 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.

The most significant improvements of previously made parts worth discussing is the improvement to the melA and crtY-crtZ (zeaxanthin) coding parts.

The melA gene is coding sequence of tyrosinase from Rhizobium etli. This tyrosinase converts tyrosine to dopaquinone. Dopaquinone is intermediate product of melanin biosynthesis pathway that will polymerizes in an enzyme-independent reaction to form melanin. Melanin production in E. coli requires supplemental tyrosine and CuSO4. The part for the melA CDS was originally made by Kazuaki Amikura of the iGEM09_Tokyo_Tech group (2009-10-14, BBa_K193600). Previously, it had been used in an expression construct using the constitutive P(lac)IQ promoter (BBa_I14032) and B0040 RBS (composite part: BBa_K193602). We have developed a different expression construct specifically for use in E. coli BL21(DE3) which takes advantage of the T7 RNA Polymerase expression construct in the DE3 cassette. This will provide tighter epxression control and help to prevent leaky expression of the tyrosinase. Additionally, the double terminator B0015 was added to increase control over the system. In order to maximize production of the tyrosinase and limit unnecessary energy expenditure by the cell, a transcriptional terminator will ensure energy is not wasted on transcribing an overly-long mRNA transcript. The melA expression construct is the improved part that we were able to characterize the most (see Results).

The crtY-crtZ parts, containing RBSs native to Pantoea ananatis had been previously developed by Chris French of the iGEM07_Edinburgh group (2007-10-24, parts BBa_I742155 and BBa_I742158). These had previously been expressed under constitutive promoters such as J23103 but not under a T7 promoter that can be used in E. coli BL21(DE3). We kept the native RBSs on these genes and incorporated it into a new part under the control of the T7 promoter (BBa_I712074) and the terminator BBa_B0015. As with the melA expression construct descried above, these new control elements will allow users to specifically control when expression starts through an IPTG induction system, as well as limit leaky expression.

 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.