Team:Glendale CC AZ/Project/PreviousResearch

From 2013.igem.org

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Revision as of 22:58, 27 August 2013

D. radiodurans - Esther

PprI- Andrew

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. coli to 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. At first, the idea of plant proteins in Deinococcus sounded unconceivable to us. However, our curiosity paid off as we discovered that Deinococcus radiodurans had homolog LEA proteins which are hypothesized to be acquired via horizontal gene transfer (Makarova et al., 2001). Three of these D. radiodurans proteins are DR0105, DR1372 and DR1172 which show similarity to well-characterized and widespread desiccation induced LEA proteins in plants (Makarova et al., 2001).

PprA- Sean

RecA- Teresa

PprM- Teresa

@@ Line 9: / Line 9: @@
 ==PprI==
-In 2012, Osaka team investigated increasing Hydrogen Peroxide 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''. In order to determine if PprI would also increase desiccation resistance in ''E. coli'', our team decided to perform a series of experiments. 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. 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 chassi, E. coli strain NBE 10β thus  [https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/AlkalineLysis 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 [https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/GrowthCurve 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_K602005 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 also ran [https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/SurivalGrowth survival plate experiments] with the same conditions and it produced similar results.
+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. coli to 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.
-PprA- Sean
-LEA- Yanet
+==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. At first, the idea of plant proteins in Deinococcus sounded unconceivable to us. However, our curiosity paid off as we discovered that Deinococcus radiodurans had homolog LEA proteins which are hypothesized to be acquired via horizontal gene transfer (Makarova et al., 2001). Three of these D. radiodurans proteins are DR0105, DR1372 and DR1172 which show similarity to well-characterized and widespread desiccation induced LEA proteins in plants (Makarova et al., 2001).
+PprA- Sean
 RecA- Teresa
 PprM- Teresa