Team:Glendale CC AZ/Project/Background/Overview

Glendale Community College Arizona

Overview

---

Previous Research

We began our project with a plan to characterize existing biobricks, as well as create a few of our own to complete a comprehensive desiccation toolkit that can be used for a variety of applications. Osaka’s 2011 iGEM team created coding device biobricks for genes PprI (BBa_K602000), PprM (BBa_K602002), PprA (BBa_K602001), and RecA (BBa_K602003) as well as expression parts for PprI (BBa_K602005), PprM (BBa_K602007), and PprA (BBa_K602006). We wanted to characterize these parts in a novel way, as Osaka did not expose their transformed bacteria to salt stress. We also planned to expose the transformed bacteria to our Hydrogen Peroxide Growth Curve Assay for further characterization. University College London’s 2012 iGEM team created a coding part for IrrE (BBa_K729001), as well as an expression device containing their coding sequence for IrrE (BBa_K729005). We planned to further characterize their IrrE biobricks by exposing the transformed E.coli to our Salt Stress Growth Curve Assay as well as our Hydrogen Peroxide Growth Curve Assay. Valencia’s 2010 team created an expression part for one of the Late Embryogenesis Abundant (LEA) proteins, which also is said to confer salt and oxidative resistance when transformed in E.coli. Our goal was to further characterize their LEA biobrick. Through rigorous research using bioinformatics, we found that homologs to these genes exist in a local bacterium, Deinococcus hopiensis. Our plan was to create coding devices and expression devices for PprI, PprM, PprA, RecA, IrrE, and LEA from the local D. hopiensis bacterium as well as a LEA biobrick from Deinococcus radiodurans. We wished to transform these genes from D. hopiensis and D. radiodurans to E.coli, as E.coli is a well-known, easily accessible, and commonly used chassis. We, would then expose the transformed bacteria to salt stress and hydrogen peroxide stress, and carefully characterize the effects.

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.

PprI

For the team’s iGEM project, we wanted to increase E. coli’s desiccation resistance to help it survive in a desert environment. To do this, we planned to insert different genes from the Deinococcus species into the E. colito increase its resistance to dessication. One of the genes our iGEM group wanted to investigate was PprI. PprI is the transcriptional regulator involved in the expression of many of the DNA damage response proteins in Deinococcus radiodurans. In 2012, the Osaka team transformed E. coli with PprI to determine its effects on hydrogen peroxide resistance. Because of this, a PprI part from this team was in the iGEM parts database. This allowed us to order their PprI part from the iGEM registry and perform our experiments with it.

LeA

During our research for desiccation-resistance factors, we came across with the 2010 Valencia iGEM project. They demonstrated that late embryogenesis abundant proteins (LEA) from soy beans provided protection against extreme temperatures, when expressed in E. coli. Since high temperatures might lead to desiccation, we decided to explore LEA proteins in more detail. Previous studies have reported that LEA protein enhances the tolerance to various environmental stresses in organisms (Liu, Zheng, Zhang, Wang, & Li, 2010). They were first discovered in cottonseed and other plants vegetative tissues that were exposed to cold temperatures, drought, and high salinity (Liu et al., 2010).

LEA proteins are part of an extensive multi-gene family and are classified based on their sequences and expression patterns (Liu et al., 2010). Since Deinococcus was our organism of interest, we looked for studies that had reported homolog LEA proteins in this bacterium.

PprA

In 2012, Osaka team investigated increasing hydrogen peroxide resistance in E. coli by transforming the bacteria with PprA, a pleiotropic protein that promotes DNA repair in Deinococcus radiodurans. In order to determine if PprA would also increase desiccation resistance in E. coli, our team decided to perform a series of experiments. To begin the project, the PprA expression device was ordered from the iGEM parts registry. This biobrick, identified as part BBa_K602006, features LacI constitutive promoter (R0010), transcriptional regulator PprA (K602001) and ribosome binding site (B0034). In addition to the PprA expression device, our team had other candidates biobricks for our experiments that were ordered at the same time. 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 37ºC. The next step was to isolate the plasmid from the chassis, E. coli strain NBE 10 β thus, a miniprep was performed. The products were run on a flash gel (link to data) verifying that we had the correct plasmid. After the verification step, we designed growth curve experiments using sodium chloride (NaCl) as the DNA damaging agent (link to data). For these growth experiments, we used liquid cultures that were inoculated with cells from the agar stabs received from iGEM. The experimental group was E. coli transformed with BBa_K602006 while the control group was the same strain of E. coli but transformed with BBa_K602003. Registry part BBa_K602003 was used as the control group because it features only the coding sequence of RecA, a component of the DNA double-strand break repair mechanism in Deinococcus radiodurans. Since the plasmid had IPTG-induced promoter, cultures containing no IPTG were used as control groups as well. In addition to growth curve experiments, we later used isolated DNA from D. hopiensis in order to see the differences in the gene between species. We designed primers for PCR to amplify the PprA gene in D. hopiensis. We later ran these PCR products on a flash gel to confirm if we had successfully amplified the gene.

---

References

• Liu, Y., Zheng, Y., Zhang, Y., Wang, W., & Li, R. (2010, May 1). Soybean PM2 protein (LEA3) confers the tolerance of Escherichia coli and stabilization of enzyme activity under diverse stresses. Current Microbiology, 60(5), 373-378. doi:10.1007/s00284-009-9552-2
• MAKAROVA, K. S., ARAVIND, L., WOLF, Y. I., TATUSOV, R., MINTON, K. W., KOONIN, E. V., & DALY, M. J. (2001, March). Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 65(1), 44-79. doi:10.1128/MMBR.65.1.44–79.2001