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Team:Purdue/The Solution/Gene Design
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<p>The killswitch was included as a safety precaution so that the modified bacteria could be easily killed. We wanted to include the kill switch because the modified bacteria are intended to be released into fields, not kept in a confined environment like a laboratory. With a kill switch, the bacteria could still be killed out in the field if they were not working as expected or if they spread farther than desired. Finding a promoter for the kill switch was complicated by the fact that most kill switches rely on things such as dramatically altering the pH or exposing the bacteria to red light, but this project required something that could get to the bacteria underground and is economically feasible to spread across an area as big as a field while not harming native plants or the natural soil microbiome. Lactose was chosen because it fulfills all of these requirements and would not work as a food source for the Bacillus subtilis.[xvii] The holin Hyb5 was selected because it is effective on Bacillus subtilis at neutral pH around field temperature and had fewer base pairs than its counterpart, the endolysin Lyb5[xviii].</p> | <p>The killswitch was included as a safety precaution so that the modified bacteria could be easily killed. We wanted to include the kill switch because the modified bacteria are intended to be released into fields, not kept in a confined environment like a laboratory. With a kill switch, the bacteria could still be killed out in the field if they were not working as expected or if they spread farther than desired. Finding a promoter for the kill switch was complicated by the fact that most kill switches rely on things such as dramatically altering the pH or exposing the bacteria to red light, but this project required something that could get to the bacteria underground and is economically feasible to spread across an area as big as a field while not harming native plants or the natural soil microbiome. Lactose was chosen because it fulfills all of these requirements and would not work as a food source for the Bacillus subtilis.[xvii] The holin Hyb5 was selected because it is effective on Bacillus subtilis at neutral pH around field temperature and had fewer base pairs than its counterpart, the endolysin Lyb5[xviii].</p> | ||
Revision as of 04:39, 17 October 2014
Our plasmids encode the enzymes necessary for the biochemical synthesis of phytosiderophores.[i] Two plasmids were used because the number of DNA base pairs for all of the genes (9,535 base pairs) was too high to fit on a single plasmid. The genes were split to make the plasmids as equal in length as possible while maintaining an appropriate configuration. This may lead to different expression of the genes on each respective plasmid, but we chose to take this risk and compensate for it in our characterization. Sequences for our enzymes were obtained from corn and codon optimized for Bacillus subtilis. The first plasmid had 4,832 base pairs and included two of the corn genes involved in the biosynthesis of phytosiderophores, the corn gene responsible for transporting phytosiderophores out of the cell, and a reporter. It starts with a constitutive promoter (“Pveg”) which has a medium level of gene expression in Bacillus subtilis[ii]. Following the promoter is an RBS; the RBS’s used in this plasmid and the second were all “SpoVG RBS”, which has been shown to have high levels of translation in Bacillus subtilis[iii]. The corn genes are naat1 (1566 base pairs), which encodes nicotianamine aminotransferase[iv], DMAS (942 bp), which encodes deoxymugineic acid synthase[v], and TOM1 (1431 bp), a transporter of mugineic acid family phytosiderophores[vi]. Following them is a reporter, sfGFP, or superfolder Green Fluorescent Protein, which has been documented as extremely effective in Bacillus subtilis (720 bp)[vii]. Following the sfGFP is a terminator (39 bp). The second plasmid had 4,703 base pairs and included the kill switch, two of the corn genes involved in the biosynthesis of phytosiderophores, and a reporter. It starts with a promoter induced by lactose (“PLlac0”, 55 bp). Following the lactose promoter is Hyb5, a holin which causes lysing [cell-death] in Bacillus subtilis (465 bp)[viii]. A terminator concludes the kill switch section of the plasmid. Following the kill switch is the constitutive promoter Pveg, the corn genes SAM (1689 bp), which encodes S-adenosylmethionine synthase,[ix] and nas1 (984 bp), which encodes nicotianamine synthase,[x] a reporter mRFP (mutant red fluorescent protein, 706 bp)[xi] which has been successfully used in Bacillus subtilis before, and a concluding terminator.
Plasmid Design
The plasmids were designed strategically so that they were roughly equal in number of base pairs, and the orders of specific were considered carefully. Shorter plasmids are more likely to be successful and cheaper, which is why we tried to minimize the length of each plasmid. Terminators are not always effective, so the kill switch is located at the beginning of a plasmid to avoid being accidently translated by an mRNA due to a previous section not being properly terminated. The reporters are placed after the corn genes because gene expression decreases linearly as the distance from the promoter increases, and expressing the corn genes is much more important than expressing the reporters.
Organization
Using a conditional promoter so that the modified Bacillus subtilis would only produce phytosiderophores in the presence of plants roots was considered, but it was decided that conditional promoters would not be reliable enough. A constitutive promoter of medium strength was chosen for the corn genes because a weak promoter might not have produced enough phytosiderophores to make a difference, and a strong promoter might have overtaxed the cell in the production of phytosiderophores as to be counterproductive. A strong RBS was chosen so that when a promoter was induced, the RBS would not be the weak link. This particular RBS was selected as a strong one because it has been shown to have high levels of translation specifically in Bacillus subtilis,[xii] even though many parts are E. coli specific and do not work well, if at all, in Bacillus subtilis. sfGFP and mRFP were chosen as the reporters because they both are expressed in Bacillus subtilis (research revealed that RFP is not expressed in Bacillus subtilis and thus should not be used with it),[xiii] visible reporters are easiest to observe, and fluorescent proteins in particular are easiest of the visible reporters, especially since the bacteria would be in a dark soil medium. Two reporters were chosen instead of using the same one over so that it could be observed whether one or both plasmids were being expressed. A relatively strong forward terminator with few base pairs was selected for its relative effectiveness, the fact that no reverse termination was required, and the number of base pairs was to be minimized as much as possible without impairing function.
Registry Part Selection
