Genes of the Color Pathway
The enzymes necessary to produce colored Escherichia coli colonies will be isolated from E. coli genomic DNA and Saccharomyces cerevisiae cDNA. A total of eight enzymes are needed to produce FPP, for the rest of the pathway we will make use of BioBrick I742152 and I742161 to make sure colonies will turn red. Other colors (orange and yellow also see the Edinburgh 2007 wiki) can be produced by adding other enzymes from the registry. Of the eight enzymes we will isolate three are E. coli enzymes (atoB, idi and ispA), while the other five are S. cerevisiae enzymes (ERG8, ERG12, ERG13, MVD1 and HMG2).
To see all parts we are working on click here. All parts with number BBa_K115050 and higher are the parts involved in color synthesis.
To PCR the genes out of the E. coli genome, we've designed primers using the invitrogen website. These primers don't contain the biobrick prefix and suffix yet. When the first PCR on the genome has worked with Taq polymerase, we will try it with Pfx polymerase which has proofreading and is more reliable. On these products, we'll perform a new PCR, with attached biobrick prefix and suffix. This way we prevent interference of the prefix or suffix in our genomic PCR. Table 1 gives an overview of the sequences of all primers designed.
|Target gene (name)||Sequence|
As we need to drain FPP constantly (FPP is toxic to the cell in high concentrations), the idea is to produce a color or the direct precursor (Phytoene) at all temperatures. We want to express the enzymes of the FPP producing pathway constitutively, independent of temperature. There is a need however to 'tune' this expression to the consumption of the color pathway. In practice this means the eight enzymes necessary for FPP production will be expressed in one operon under the same promotor. However, the required strength of the promotor and ribosome binding site will have to be determined experimentally. The color enzymes (from FPP to lycopene, B-carotene and zeaxanthin) will be expressed seperately and under regulation of the RNA thermometers. As we will obtain three colors, this would mean two switches, for instance at 27ºC and 37ºC in the case of constitutively induced lycopene production. This means red colonies will form at all temperatures below 27ºC. However the enzyme for B-carotene production would be switched on (by loss of secondary structure of the RNA element) at 27ºC and between 27ºC and 37ºC E. coli colonies will be orange. Above 37ºC the zeaxanthin enzyme will be turned on and colonies will have a yellow color. If the basic molecule will be phytoene, there is room for three RNA thermometers, for example 27ºC, 32ºC and 37ºC. This would give the temperature scheme of:
- T < 27ºC: 'E. coli color'
- 27ºC < T < 32ºC: Red
- 32ºC < T < 37ºC: Orange
- 37ºC < T : Yellow
The first gradient PCR have been performed on the genes, the E. coli genes all worked correctly (see lab notebook entry of August 19th), while still some work has to be done on the S. cerevisiae primers (August 20th). The next step will be to PCR the E. coli genes again, with the ideal annealing temperature, using Pfx polymerase instead of Taq. The Pfx enzyme has proofreading and so is less likely to make a mistake within the genes. The gradient PCR on S. cerevisiae cDNA will be repeated and optimized.