I gem 2012 design competition design team 4 proposal

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IGEM final design report

IGEM final design report

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  • 1. Michael PettigrewHansika SarathchandranAbby PollockWendy XiangSharon RavindraniGEM Team 4 Design Proposal - Application of Li16 to Crop PreservationIntroduction and SignificanceThis proposal focuses on the novel gene li16 and its associated protein expressed in theliver of the common wood frog (Rana sylvatica). Significant up-regulation of li16 has beenidentified in response to freezing providing a strong linkage to a role in freezing survival,Storey et al [1]. Such anti-freeze proteins (AFPs) is of significant interest for their effect infreezing prevention and modern applications in organ transplant, cryoprotection, andproduction of frozen foods. The Yale iGem team conducted a related project in 2011 titled“Nature’s Antifreeze” where they expressed and characterized an antifreeze protein fromthe Siberian beetle, Rhagium inquisitor (RiAFP) [2]. It was found that RiAFP inhibited icerecrystallization in a dose-dependent manner; and post-freezing survival was improved forE. coli expressing the protein. Building upon Yale’s project but taking a different approach,the team plans to use the protein for applications in crop preservation, and if possible organpreservation. Freeze/frost damage to crops are of significant concern to the agriculturalindustry, making improved crop preservation in freezing temperatures a worthwhile goal topursue.The mechanisms involved in the proposed project consists of several distinct sections. Theli16 must first be isolated then incorporated into a biobrick part to enable expression of theli 16 protein in E. Coli. Responses to freezing will be analyzed using ice recrystallizationassays and freeze tolerance tests. Finally, the protein will be isolated and applied to targetcrops in order to determine and characterize any effects on crop preservation in freezingenvironments.Background:Many cold-blooded animals use biochemical adaptations to adapt to freezingenvironments, especially by preventing the freezing of body fluids. The wood frog (Ranasylvatica) is a well studied species for their freeze tolerance characteristics and theassociated antifreeze proteins. So far there have been six freeze up-regulated genesidentified in this species. The most recently identified fr47 codes for a protein expressed inthe liver of these frogs. Others are fr10 (expressed in all organs), li16 (expressed liver, heart,
  • 2. and gut), mitochondrial inorganic phosphate carrier (PiC), NADH-ubiquinoneoxidoreductase subunit 4 (ND4), and elongation factor 1 gamma subunit (EF-1).We will be specifically working with the protein Li16, as recent studies have hypothesizedthat it plays a significant role in freeze response. In addition, analysis has indicated that Li16is not similar to any currently known proteins, unlike the other freeze up-regulated proteinsdescribed. This presents an opportunity for novel and important work.All previous work on the Li16 protein has been performed by the Storey lab [reference lab].This lab recently examined Li16 expression in 12 types of wood frog tissue. Li16 was firstdiscovered the liver of the frog. The li16 transcript codes a protein with 115 amino acids, ofwhich 16 are basic and 11 are acidic, with an overall pH of 8.29. [1] The protein has acalculated molecular mass of 12.8 kDA.[1] From the testing of anoxia and dehydrationconditions over different tissues, numerous results were discovered. In previous reportsdone by the Storey lab they had used Northern blot but to increase the analysis of Li16 theyused an RT- PCR which provided a sensitive analysis. The first result was that after a 24freezing period, the amount of li16 transcripts increased by 3 folds in testes, 2 folds inheart, ventral skin, lung. [1] The increase of li16 transcripts in the brain, liver, heartdemonstrated a direct relationship in the increase of Li16 protein. The increase in the levelof li16 transcripts was frequently mirrored by the increase of the li16 protein. The Storeylab concluded that through the increase of Li16 and the fact that li16 transcript were foundin all 12 tissues tested and Li16 protein was found in all 6 tissues tested indicates that Li16played a role in freezing.[1] Unusual results that were obtained included the level of Li16showed no significant change in the liver and the level of Li16 appeared to drop in thedorsal skin. In addition, the greatest increase of Li16 was found in ventral skin. The fact thatventral skin is the one that is closest to the environment and a thin tissue would make it themost vulnerable to freezing. Therefore, the transcription and translation must occur rapidlyfor protein synthesis which correlates with the result of ventral skin being one of the tissuesthat produced a high level of Li16 demonstrating the significance Li16 proteins in freezing.Obtaining Li16Li16 is currently not present as a biobrick in the parts registry. In order to create this part, itis first necessary to obtain the Li16 gene. To do this, we can either (1) contact the Storey laband request a cDNA copy of the gene, (2) isolate a cDNA copy of the gene from frog tissueusing RT-PCR, or (3) have the Li16 cDNA created by a gene synthesis company. Once thegene has been obtained, it can be turned into a biobrick part through PCR, using primerswhich contain the biobrick prefix and suffix and a short sequence found on the Li16 gene.This is detailed here: http://partsregistry.org/Help:Primers/Design.Design
  • 3. Once the Li16 biobrick part is created, it will be utilized in a series of standard constructionsto turn it into a functional translational unit plus promoter. The promoter used will beinducible so that we can measure the response to the protein as a function of proteinconcentration.TestingThis system will then be expressed in E. coli, and their response to freezing will bemeasured. These responses will be measured through ice recrystallization inhibition assaysand freeze tolerance tests. Ice recrystallization induces cell injury, therefore, inhibition ofice recrystallization becomes a positive indicator improved cryoprotection. The type ofrecrystallization inhibition assay that was used to view the ice recrystallization inhibitionwas a modified splat assay. Within the modified splat assay, the sample was sandwichedwith glass slides and a sucrose solution, afterwards the assay was cooled and imaged atvarious time intervals. [2] For the freeze tolerance tests, we will determine viability of E. coliafter they are frozen for a certain amount of time for E. coli expressing different levels ofLi16, as compared to a control. In addition, we will measure bacterial responses to anoxiaand dehydration, as these conditions are closely related to freezing.Once the system has been expressed in E. coli and various freeze tolerance tests have beenperformed, we will isolate the Li16 protein so that futher experimentation can beperformed on it.Based on the assumption that the target protein is an antifreeze protein (AFP) the iceaffinity purification method can be used to purify Li16 from the E. coli. Yale also used thismethod in their 2011 project. This method grows layers of ice on a cold finger in a solutionof crude cell lysate, purifying the protein based on the its property to selectively bind togrowing ice crystals [3].This isolated protein will then be applied to the application of crop preservation. Methodsof application of Li16 to crops can range from root uptake to spraying, followed by freezetreatments (range from -5 degrees Celsius to -30 degrees) with a control group. Differential
  • 4. thermal analysis (DTA) and nuclear magnetic resonance spectroscopy (NMR) that measurestemperature and volume of liquid water respectively can be used to evaluate and comparedegrees of freezing [4]. (Different types of plants have been used to test the effectiveness oftreatments, e.g. Arabidopsis plants can be used for their high availability.)Expected ResultsThe input to the system will be anhydrotetracycline (aTc). We expect a linear relationshipbetween aTc concentration in the bacterial media, and the freeze tolerance of the E. coli.We expect that bacteria without the Li16 system will have a constat freeze tolerance,independent of the concentration of aTc.Feasibility of Design, Problems, and ContingenciesThe first challenge encountered will be obtaining the Li16 protein. As aforementioned, Li16is currently not present as a biobrick in the parts registry. While it would be ideal to get acopy of the gene from the Storey lab, this may not be possible. Isolating the gene ourselveswould be time consuming and difficult, as we would need to obtain the cDNA due toprobable introns in the Li16 sequence. Our other option of having it created by a genesynthesis company, would save us time but would be expensive.Once the gene has been obtained, it should not be difficult to convert into a biobrick part.As well, creating the overall system with promoter, RBS, and terminator should bestraightforward as all of these parts have been used often and are well characterized in theregistry.The main difficulty associated with this project begins when we transform the E. coli withthe plasmid containing the Li16. It is possible that the Li16 could not fold properly without‘helper’ proteins only present in eukaryotes. In addition, the Li16 may be toxic to thebacteria. If this is the case, there are various strategies that can be tried. These include
  • 5. creation of a fusion protein with, for example, GFP; codon optimization so that the proteinis more easily translated by the bacteria; identification of a function domin of Li16, and thenfusion of this domain to a protein such as GFP.ConclusionsAs previously established, the report expands on Yale’s project by focusing on one specificprotein, Li16 and observing the effect that this protein had on the tissues of an organism.From the report done by the Storey lab, it was concluded that Li16 plays a significant role,which was especially seen through the dramatic increase in protein in the tissue after a 24hour freezing period. Through the creation of the Li16 biobrick, it will be expressed in Ecoli,where the antifreeze protein will be isolated. The antifreeze protein isolation is the mainobjective of this report, which will be used to prevent the freezing of crops in suddenweather changes and hence preserving the life of the crops.References[1] Storey and Sullivan. 2012. Environmental stress responsive expression of the gene li16 inRana sylvatica, the freeze tolerant wood frog. Cryobiology.[2] Yale iGem. 2011. Nature’s Antifreeze: Microbial Expression and Characterization of a NovelInsect Antifreeze Protein for De-icing Solution. Internet: http://2011.igem.org/Team:Yales[3] Peter L. Davies. Antifreeze Proteins. Internet:http://pldserver1.biochem.queensu.ca/afp/afp.shtml[4] Rogers S. Pearce. 2001. Plant freezing and damage. Internet:http://aob.oxfordjournals.org/content/87/4/417.full.pdf