"
Team:Glendale CC AZ/Project
From 2013.igem.org
(→Experiments) |
|||
| Line 41: | Line 41: | ||
PprI | PprI | ||
| - | In 2011, Osaka team reported success increasing ionizing radiation resistance in ''E. coli'' by transforming the bacteria with PprI, the transcriptional regulator involved in the expression of many DNA damage response proteins in ''Deinococcus radiodurans''. Our team decided to test PprI in order to determine if it would also increase desiccation resistance in ''E. coli''. To begin the project, the PprI expression device was ordered from the IGEM parts registry. This biobrick, identified as part BBa_K602005, features LacI constitutive promoter (R0010), transcriptional regulator PprI (K602000) and ribosome binding site (B0034). In addition to PprI, our team had other candidates biobricks for our experiments that were ordered at the same time as PprI. We received all the biobricks in the form of agar stabs that were inoculated into LB/chloramphenicol liquid media. The liquid cultures were incubated overnight at 37º C. The next step was to isolate the plasmid from the cells, an alkaline lysis plasmid miniprep was performed with samples taken from the cultures. The products from that miniprep were run on a flash gel verifying that we had the correct plasmids. After isolating the PprI plasmid, growth curves using the cells from the agar stab were performed. In these experiments, Rec A, registry part BBa K602003, was used as a control. This is because the Rec A cells had only the coding sequence, no promoter, so Rec A would not have been expressed. The conditions for each of the tubes used in the growth curves is as follows: | + | In 2011, Osaka team reported success increasing ionizing radiation resistance in ''E. coli'' by transforming the bacteria with PprI, the transcriptional regulator involved in the expression of many DNA damage response proteins in ''Deinococcus radiodurans''. Our team decided to test PprI in order to determine if it would also increase desiccation resistance in ''E. coli''. To begin the project, the PprI expression device was ordered from the IGEM parts registry. This biobrick, identified as part BBa_K602005, features LacI constitutive promoter (R0010), transcriptional regulator PprI (K602000) and ribosome binding site (B0034). In addition to the PprI expression device, our team had other candidates biobricks for our experiments that were ordered at the same time as PprI. We received all the biobricks in the form of agar stabs that were inoculated into LB/chloramphenicol liquid media. The liquid cultures were incubated overnight at 37º C. The next step was to isolate the plasmid from the cells, an alkaline lysis plasmid miniprep was performed with samples taken from the cultures. The products from that miniprep were run on a flash gel verifying that we had the correct plasmids. After isolating the PprI plasmid, growth curves using the cells from the agar stab were performed. In these experiments, Rec A, registry part BBa K602003, was used as a control. This is because the Rec A cells had only the coding sequence, no promoter, so Rec A would not have been expressed. The conditions for each of the tubes used in the growth curves is as follows: |
1 | 1 | ||
Revision as of 22:26, 26 July 2013
| Home | Team | Official Team Profile | Project | Submitted Parts | Protocols | Notebook | Safety | Attributions | Human Practices |
|---|
Contents |
Background
Overview
Desert areas, making up almost one-quarter of the Earth surface, are home to 500 million people. As a result of human habitation, every continent in the world except Antarctica is increasingly and adversely affected by desertification. Studies forecasting climate trends indicate that desert regions will face an even drier future – in regard to both climatic factors and drought conditions – stemming from the influence of human activities. One human-created impact on these desert environments is pollution. Many remediation challenges exist specific to the extremely dry conditions present in these arid locales. For example, what happens when a desert area is polluted with such substances as plastics, where commonly employed bioremediation agents used for environmental cleanup cannot survive desert climates’ high temperature and low humidity extremes? Our team has developed a kit containing biological components that will provide desiccation resistance to organisms used to facilitate the elimination of contaminants, like plastics, in desert-like environments. Our goal is to design an assortment of complementary parts, which will strengthen organisms and help to broaden their climatic and geographic range of effectiveness. While some parts included in our kit could, conceivably, provide resistance to other types of stresses, such as ionizing radiation, we will initially focus on desiccation. Because of its potential to expand the extent of the efficacy of these biological breakdown expedients into even climatically extreme territories, our kit could be a valuable addition to any bioremediation project.
Growing Desert
Desert regions are currently found scattered around the globe, but, with the advent of global warming, both the boundaries of these regions and the appearance of new areas that could be seen as “desert-like” are increasing at a rapid rate. Rainfall is predicted to decrease by as much as 20% by the end of the century, causing water sources to be depleted and wells to run dry, ultimately resulting in the death of plants and animals. Consequently, humans will migrate to more climatically hospitable locations, which, in short order, has the likelihood of creating the very same problems in these newly habited areas not previously impacted by desertification, thus perpetuating a vicious cycle of depletion, desiccation, and death. Irrigation used for agriculture may, in the long term, lead to soil with salinity levels too high to support plants. Higher temperatures can also produce an increasing number of wildfires, which alter desert landscapes through the elimination of slow-growing trees and shrubs, followed by an influx of highly flammable fuel in the form of fast-growing grasses. In addition, as glaciers, which provide a large portion of the water used for agricultural and domestic purposes in the deserts of the southwestern United States, South America, and Central Asia, continue to melt, the loss of these ancient sources threatens to further limit water availability in these regions. Glaciers keep melting which threatens water availability in some areas.
Threatened Desert
Wet Lab
Overview
In the past other teams (Osaka, University College London) have explored the resistance genes in the bacteria D. radiodurans. These resistance genes convey resilience against high levels of radiation, oxidative stress and desiccation in that the DNA repairs itself when damaged from these stressors. The interesting thing about these stressors is that the DNA repairs itself in the same way regardless of the type of stress. Simply put, the system responds to DNA damage and makes the necessary repairs. From here, the project first aims to provide extra validation data to those studies. Additionally the ultimate purpose of this iGEM project is to explore the resistance genes in a similar bacteria, Deinococcus hopiensis, as a novel source of these genes within the Deinococcus genus. For this purpose the project then uses the genes to transform a desiccation, radiation, and oxidative stress sensitive bacteria, E. coli to improve the robustness of the cells against the particular stressors of desiccation and oxidative stress. In science, adding validation data is always a very important and welcome resource. In addition to the validation data, the main goal of the project is to look for these same genes in a sister bacteria and see if those genes convey the same resistance.
Experiments
PprI
In 2011, Osaka team reported success increasing ionizing radiation resistance in E. coli by transforming the bacteria with PprI, the transcriptional regulator involved in the expression of many DNA damage response proteins in Deinococcus radiodurans. Our team decided to test PprI in order to determine if it would also increase desiccation resistance in E. coli. To begin the project, the PprI expression device was ordered from the IGEM parts registry. This biobrick, identified as part BBa_K602005, features LacI constitutive promoter (R0010), transcriptional regulator PprI (K602000) and ribosome binding site (B0034). In addition to the PprI expression device, our team had other candidates biobricks for our experiments that were ordered at the same time as PprI. We received all the biobricks in the form of agar stabs that were inoculated into LB/chloramphenicol liquid media. The liquid cultures were incubated overnight at 37º C. The next step was to isolate the plasmid from the cells, an alkaline lysis plasmid miniprep was performed with samples taken from the cultures. The products from that miniprep were run on a flash gel verifying that we had the correct plasmids. After isolating the PprI plasmid, growth curves using the cells from the agar stab were performed. In these experiments, Rec A, registry part BBa K602003, was used as a control. This is because the Rec A cells had only the coding sequence, no promoter, so Rec A would not have been expressed. The conditions for each of the tubes used in the growth curves is as follows:
1 2 3 4 5 6 7 8 9
In this experiment, nacl was needed because it tests the desication resistance of our bacteria. In addition, IPTG was needed because the plasmid the PprI gene was on had an IPTG induced promoter, which means PprI wouldn't be expressed unless there was IPTG in the solution.
