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Team:Tsinghua-A/Project
From 2014.igem.org
1
Overview
What is TAL effector?
TAL (transcription activator-like) effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species. These proteins are able to bind promoter sequences in the host plant and to activate the expression of plant genes that aid bacterial infection.[i]
What can TAL effectors do and how do they function?
There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence. [ii]As a result, TAL effectors have attracted great interest as DNA targeting tools.
Their targeting specificity is determined by a central domain of tandem, 33–35 amino acid repeats, followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats. A polymorphic pair of adjacent residues at positions 12 and 13 in each repeat, the ‘repeat-variable di-residue’ (RVD), specifies the target, one RVD to one nucleotide, with the four most common RVDs each preferentially associating with one of the four bases.[iii][iv]
Figure 1. Structure of a naturally occurring TAL effector[v]
What’s the weakness of TAL effector?
This simple code between amino acids in TAL effectors and DNA bases in their target sites might be useful for protein engineering applications. Numerous groups have designed artificial TAL effectors capable of recognizing new DNA sequences in a variety of experimental systems. It has been reported that TAL effectors target genes efficiently in many eukaryocytic cells like mammalian cells and yeasts. However, the former experiments with E. coli indicate that TAL effectors don’t work well in E. coli cells.
References
[i] Bogdanove AJ, Schornack S, Lahaye T. TAL effectors: finding plant genes for disease and defense. Curr. Opin. Plant Biol. 2010; 13: 394-401.
[ii] Boch J, Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu. Rev. Phytopathol. 2010; 48: 419-436.
[iii] Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science. 2009; 326: 1501.
[iv] Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 2009; 326:1509-1512.
[v] Tomas Cermak, Adam J. Bogdanove, and Daniel F. Voytas. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucl. Acids Res. 2011; 39: 7879-7879
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Hypothesis
It has been reported that TAL effectors target genes efficiently in many eukaryocytes like mammalian cells and yeasts. However, the former experiments with E. coli indicate that TAL effectors don’t work well in E. coli cells. There are a variety of factors can possibly be attributed to its low efficiency, and homologous recombination is a highly possible one.
Some proteins originated from prokaryocytes, with their sequences paralleling with DNA sequences of E. coli, their expression might be hampered because of homologous recombination. By parity of reasoning, we propose a hypothesis that the inefficiency of TAL effector expression is caused by its sequence resemblance with E. coli genes.
Our solution is based on the codon usage bias, which refers to differences in the frequency of occurrence of synonymous codons in coding DNA.[1] The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism.[2]
Figure 1. RNA codon table[3]
References
[1] Susanta K. Behura* and David W. Severson, Codon usage bias: causative factors, quantification methods and genome-wide patterns: with emphasis on insect genomes. Biological Review. 2012; 88: 49-61
[2] Lanza, Amanda M.; Curran, Kathleen A.; Rey, Lindsey G.; et al. A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae. BMC Systems Biology. 2014; 8: 33.
[3] http://bioinfo.bisr.res.in/cgi-bin/project/crat/theory_codon_restriction.cgi
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TALE Assembly
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TALE Expression
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3
Report System
We construct a report system so as to test the reliability and efficiency of our ‘Marvelous TALE’. In this section, we test the TALE’s DNA binding ability and report it with a common report gene ‘RFP’. We attempt to put the target of TALE’s DNA binding target sequence inside the expression cassette of report gene and binding TALE can disrupt the express of report gene. We use iGEM standard parts to build our report system.
We designed a standard iGEM part BBa_K1311007 to complete all the tasks. This part contains
Promoter (J23102)-TALE binding site (repeats three times)-RBS-LacI coding sequence-Terminator(B0015)-LacI Regulative Promoter(R0010)-RBS(B0034)-mRFP1(E1010)-Terminator(B0015)
This part can convert the binding ability of TALE protein to its target DNA sequence to an easier available parameter, the florescent intensity of RFP. When the TALE protein is expressed, the TALE make binds to its target, which may interrupt the transcription of LacI. The lack of repressors may lead to the expression of RFP. So the stronger florescent intensity means the better binding ability of TALE protein. In this part, the target of TALE recognition site is chosen to be the 18bp sequence (ACCTCATCAGGAACATGTT).
Our Circuit Design
This parts can convert the binding ability of TALE protein to its target DNA sequence to a easier available parameter, the florescent intensity of RFP. When the TALE protein is expressed, the TALE make binds to its target, which may interrupt the transcription of LacI. The lack of repressors may lead to the expression of RFP. So the stronger florescent intensity means the better binding ability of TALE protein.
Validation
We change the normal RBS sequence in the LacI coding sequence into an RBS containing three tandem TALE binding site sequences. So we have to validate that the LacI protein can express normally and can normally inhibit the expression of RFP.
We transformed this plasmid in to E.coli DH5α with electroporation and chemical transformation. We spread the plate and culture it in 37 degrees Centigrade for more than 15 hours until small colonies can be seen in the plate. At this moment, the colonies might look red for the sake of the delay of expression of LacI. We picked colonies into the 10mL tubes and added 5mL LB broth with Chl antibiotics.
After six hours’ shaking at 37 degrees Centigrade and 220 rpm, we got the tubes out and double, four times, eight times diluted. (40μL of 0.1M IPTG was added as the positive control group) After another shaking for 12h, florescence of the bacteria was evaluated with an enzyme-labeled instrument and OD600 was tested with spectrometry.
Picture of our tubes
(The two red ones on the back are the positive control groups, others are not red)
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About Us
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