The document proposes expressing the li16 gene from wood frogs in E. coli to study its antifreeze protein Li16. Li16 increases in wood frogs after freezing and may help freezing survival. The team will obtain li16, express it in E. coli, and test the effects on freezing using ice recrystallization assays and freeze tolerance tests. Isolated Li16 protein will then be applied to crops to study effects on freezing preservation. Challenges include obtaining li16 and ensuring proper folding in E. coli.

1. Michael Pettigrew
Hansika Sarathchandran
Abby Pollock
Wendy Xiang
Sharon Ravindran
iGEM Team 4 Design Proposal - Application of Li16 to Crop Preservation
Introduction and Significance
This proposal focuses on the novel gene li16 and its associated protein expressed in the
liver of the common wood frog (Rana sylvatica). Significant up-regulation of li16 has been
identified 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 in
freezing prevention and modern applications in organ transplant, cryoprotection, and
production 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 from
the Siberian beetle, Rhagium inquisitor (RiAFP) [2]. It was found that RiAFP inhibited ice
recrystallization in a dose-dependent manner; and post-freezing survival was improved for
E. 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 organ
preservation. Freeze/frost damage to crops are of significant concern to the agricultural
industry, making improved crop preservation in freezing temperatures a worthwhile goal to
pursue.
The mechanisms involved in the proposed project consists of several distinct sections. The
li16 must first be isolated then incorporated into a biobrick part to enable expression of the
li 16 protein in E. Coli. Responses to freezing will be analyzed using ice recrystallization
assays and freeze tolerance tests. Finally, the protein will be isolated and applied to target
crops in order to determine and characterize any effects on crop preservation in freezing
environments.
Background:
Many cold-blooded animals use biochemical adaptations to adapt to freezing
environments, especially by preventing the freezing of body fluids. The wood frog (Rana
sylvatica) is a well studied species for their freeze tolerance characteristics and the
associated antifreeze proteins. So far there have been six freeze up-regulated genes
identified in this species. The most recently identified fr47 codes for a protein expressed in
the liver of these frogs. Others are fr10 (expressed in all organs), li16 (expressed liver, heart,

2. and gut), mitochondrial inorganic phosphate carrier (PiC), NADH-ubiquinone
oxidoreductase subunit 4 (ND4), and elongation factor 1 gamma subunit (EF-1).
We will be specifically working with the protein Li16, as recent studies have hypothesized
that it plays a significant role in freeze response. In addition, analysis has indicated that Li16
is not similar to any currently known proteins, unlike the other freeze up-regulated proteins
described. 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 first
discovered the liver of the frog. The li16 transcript codes a protein with 115 amino acids, of
which 16 are basic and 11 are acidic, with an overall pH of 8.29. [1] The protein has a
calculated molecular mass of 12.8 kDA.[1] From the testing of anoxia and dehydration
conditions over different tissues, numerous results were discovered. In previous reports
done by the Storey lab they had used Northern blot but to increase the analysis of Li16 they
used an RT- PCR which provided a sensitive analysis. The first result was that after a 24
freezing period, the amount of li16 transcripts increased by 3 folds in testes, 2 folds in
heart, ventral skin, lung. [1] The increase of li16 transcripts in the brain, liver, heart
demonstrated a direct relationship in the increase of Li16 protein. The increase in the level
of li16 transcripts was frequently mirrored by the increase of the li16 protein. The Storey
lab concluded that through the increase of Li16 and the fact that li16 transcript were found
in all 12 tissues tested and Li16 protein was found in all 6 tissues tested indicates that Li16
played a role in freezing.[1] Unusual results that were obtained included the level of Li16
showed no significant change in the liver and the level of Li16 appeared to drop in the
dorsal skin. In addition, the greatest increase of Li16 was found in ventral skin. The fact that
ventral skin is the one that is closest to the environment and a thin tissue would make it the
most vulnerable to freezing. Therefore, the transcription and translation must occur rapidly
for protein synthesis which correlates with the result of ventral skin being one of the tissues
that produced a high level of Li16 demonstrating the significance Li16 proteins in freezing.
Obtaining Li16
Li16 is currently not present as a biobrick in the parts registry. In order to create this part, it
is first necessary to obtain the Li16 gene. To do this, we can either (1) contact the Storey lab
and request a cDNA copy of the gene, (2) isolate a cDNA copy of the gene from frog tissue
using RT-PCR, or (3) have the Li16 cDNA created by a gene synthesis company. Once the
gene has been obtained, it can be turned into a biobrick part through PCR, using primers
which 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 constructions
to turn it into a functional translational unit plus promoter. The promoter used will be
inducible so that we can measure the response to the protein as a function of protein
concentration.
Testing
This system will then be expressed in E. coli, and their response to freezing will be
measured. These responses will be measured through ice recrystallization inhibition assays
and freeze tolerance tests. Ice recrystallization induces cell injury, therefore, inhibition of
ice recrystallization becomes a positive indicator improved cryoprotection. The type of
recrystallization inhibition assay that was used to view the ice recrystallization inhibition
was a modified splat assay. Within the modified splat assay, the sample was sandwiched
with glass slides and a sucrose solution, afterwards the assay was cooled and imaged at
various time intervals. [2] For the freeze tolerance tests, we will determine viability of E. coli
after they are frozen for a certain amount of time for E. coli expressing different levels of
Li16, as compared to a control. In addition, we will measure bacterial responses to anoxia
and dehydration, as these conditions are closely related to freezing.
Once the system has been expressed in E. coli and various freeze tolerance tests have been
performed, we will isolate the Li16 protein so that futher experimentation can be
performed on it.
Based on the assumption that the target protein is an antifreeze protein (AFP) the ice
affinity purification method can be used to purify Li16 from the E. coli. Yale also used this
method in their 2011 project. This method grows layers of ice on a cold finger in a solution
of crude cell lysate, purifying the protein based on the its property to selectively bind to
growing ice crystals [3].
This isolated protein will then be applied to the application of crop preservation. Methods
of application of Li16 to crops can range from root uptake to spraying, followed by freeze
treatments (range from -5 degrees Celsius to -30 degrees) with a control group. Differential

4. thermal analysis (DTA) and nuclear magnetic resonance spectroscopy (NMR) that measures
temperature and volume of liquid water respectively can be used to evaluate and compare
degrees of freezing [4]. (Different types of plants have been used to test the effectiveness of
treatments, e.g. Arabidopsis plants can be used for their high availability.)
Expected Results
The input to the system will be anhydrotetracycline (aTc). We expect a linear relationship
between 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 Contingencies
The first challenge encountered will be obtaining the Li16 protein. As aforementioned, Li16
is currently not present as a biobrick in the parts registry. While it would be ideal to get a
copy of the gene from the Storey lab, this may not be possible. Isolating the gene ourselves
would be time consuming and difficult, as we would need to obtain the cDNA due to
probable introns in the Li16 sequence. Our other option of having it created by a gene
synthesis 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 be
straightforward as all of these parts have been used often and are well characterized in the
registry.
The main difficulty associated with this project begins when we transform the E. coli with
the 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 the
bacteria. 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 protein
is more easily translated by the bacteria; identification of a function domin of Li16, and then
fusion of this domain to a protein such as GFP.
Conclusions
As previously established, the report expands on Yale’s project by focusing on one specific
protein, 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 24
hour 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 main
objective of this report, which will be used to prevent the freezing of crops in sudden
weather changes and hence preserving the life of the crops.
References
[1]
Storey and Sullivan. 2012. Environmental stress responsive expression of the gene li16 in
Rana sylvatica, the freeze tolerant wood frog. Cryobiology.
[2]
Yale iGem. 2011. Nature’s Antifreeze: Microbial Expression and Characterization of a Novel
Insect 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