Difference between revisions of "Team:OUC-China/Human Practices"

Background

Nowadays，quantitative has become a general trend, and of which the control of gene expression play a vital role. Until now, there’re several ways to control gene expression and consequently achieve stoichiometry and functional protein products.

Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products(protein or RNA). Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein.

Modification of DNA(左侧插图~~~~)

In eukaryotes, the modification of DNA’s chromatin structure, such as histone modifications directed by DNA methylation[1], ncRNA, or DNA-binding protein, may up or down regulate the expression of a gene.

Regulation of transcription(左侧插图~~~~)

This controls when transcription occurs and the amount of RNA be created. The mechanisms usually includes specificity factors, general transcription factors, repressors, activators, enhancers [2] and silencers.

Post-transcriptional regulation [3] (左侧插图~~~~)

In eukaryotes, there have some mechanisms on how much the mRNA is translated into proteins [4]. Cells do this by modulating the capping, splicing, addition of a Poly(A) Tail, the sequence-specific nuclear export rates, and, in several contexts, sequestration of the RNA transcript.

Regulation of translation(左侧插图~~~~)

The translation of mRNA can also be controlled by a number of mechanisms[5], mostly at the level of initiation. The secondary structure of mRNA, antisense RNA binding, or protein binding[6] can all modulate the recruitment of the small ribosomal subunit.

So many regulation methods have developed to achieve gene expression on desired level. Accurate as they are, they can hardly control the relative expression of several cistrons simultaneously. They are usually performed on operon level and may not have difference influence on each cistron.

But on certain condition, it’s of vital importance to realize differential gene expression in polycistrons. For example, Team Imperial 2014 aimed to biosynthesize bacterial cellulose in E.coil, they need to transform two cistrons of different expression level into E.coil. Without efficient approaches, they did lots of jobs. They selected proper copied plasmid among 9 plasmids and then measured 15 Anderson promotors of different strength and finally selected a proper combination for the two different cistrons.

This year, OUC-iGEM team devoted to exploring a novel regulation method on post-transcriptional level to realize differential expression in a polycistron. Details see the design part.

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Design

Overview

In C.Cellulolyticum，it is reported that a novel regulation method was employed to regulate the differential expression of multiple cistrons within the same operon[1]. And we came out the idea of exploiting this mechanism to a toolkit using for regulating differential and quantitative expression within a polycistron.

The principle is as follows:

Figure 1. Diagram of the post-transcriptional regulation method

In bacteria genomes, genes are frequently organized as an operon that mostly encodes protein complexes, which is an efficient means to regulate transcription of multiple genes simultaneously. It is firstly transcribed by its sole promoter into the primary mRNA.
And then, in E.coli cases, the processing is initiated via a primary cleavage by RNase E[2]. It is a single-stranded, nonspecific endonuclease with preference for cleaving A/U-rich sequence and its cleavage signals locate in the intergenic regions. Primary transcript then will be split by RNase E into several secondary transcripts. [Fig.3]
Stability of these secondary transcripts varied widely due to their distinct terminal sequences that convey resistance to exonuclease degradation. In other words, stem loops with lower free energy can protect mRNA more from exoribonuclease degradation and stay longer in cells, thus the transcripts can translate more proteins.
With this system, we can regulate multiple genes in one operon express on desired quantitative level, realize multiple cistrons express coordinately, just like a melodic cistrons concerto.
To achieve this, we constructed dual-fluorescent and tri-fluorescent reporter system and designed a series of stem-loops to insert into the reporter system to test this regulating method. Following are our basal designs.

Dual-fluorescent reporter system

We want to test the protection effect on the upstream gene from stem-loops, thus we primarily construct a dual-fluorescent reporter system. The circuit is as follows:

Figure 2. Dual-fluorescent reporter

The promoter is inducible with arabinose and the two fluorescent reporter gfp and mCherry with their RBS are two parts in the iGEM part library, numbered BBa_I13500 and BBa_J06602, the RNase E site between them is from E.coli pap operon [3], a relative high efficient endonuclease cleavage site through experiments. The stem-loops are from the series of stem loops we designed.

Stem-loops

We designed a series of stem-loops of gradient free energy to test this regulation method. And we determined the free energy of stem-loops with RNA folding program (Mfold). [4][5]

We designed stem-loops in the view of 3 parts: loop, stem and a tail ahead with a pair of CG as "sealing nucleotides", see as figure 3.

Figure 3: Diagram of the designed stem-loops

Loop: Since the loop makes little contribution to the whole free energy, we used the same loop throughout our designs. That is U-U-A-C-A-U-G-A-U-U.
Stem: The stem-loop's free energy is mainly decided by the pairing stem section. We chose the ratio of "A+U" to "C+G" as 1:1. For too many C/G may cause the energy too low and hard to adjust, whereas too many A/U are easy to cause terminating effect owing to the structure similarity to rho-independent terminators[6].. Choosing the ratio as 1:1, we can easily adjust the free energy by change the length of the paring stem.
"Sealing nucleotides": A pair of CG at the end of a stem-loop can help stabilizing the secondary structure by avoiding flanking extra base paring in different context circuit.
Following these principles, the stem-loops we designed are diaplayed in table 1, they are of gradient free energy:

We measured these stem loops in the dual-fluorescent reporter system. By analyzing the amount of mRNA and protein expressed in different circuits, we aimed to explore the correlation between the folding free energy and the quantitative expression. We measured the quantitative expression on two levels: the transcriptional level and the translational level. On the transcriptional level, we aimed to find if the difference is actually caused by the varied mRNA degradation. Upon the translational level, we explored the possibility and prospect of this post-transcriptional regulation mechanism on practical application.

Tri-fluorescent reporter system

By the dual-fluorescent reporter system we obtain the correlation between folding free energy and quantitative expression of mRNA and protein fluorescence. Then we constructed a tri-fluorescent reporter system to furtherly test this relationship. The circuit is as follows:

Figure 4

We use tri-fluorescent reporters to test the correlation we obtained, the inserted CFP with RBS is also a part in the part library, numbered BBa_I13600.
We use this circuit to measure the effect of two different stem loops, with folding free energy of -51.4kcal/mol and -25.6kcal/mol. Detailed data see our result page.

Future

This year, our team devoted to exploring a novel regulation method on the post-transcription level. Once the correlation is established and confirmed, we can apply this to tune the expression of polycistron of interest. To achieve our goals, we have 3 steps to go:

a)Through our data analysis and modelling get the correlation. This step is finished in our lab with the basal circuit, the constructed fluorescent reporter system.
b)To apply this regulation method to practice using, we will develop it as a toolkit. Now we are exploiting a software to realize structure prediction base on the different folding free energy according to the target ratio.
c)Then we can employ our software a specific protein for expression optimizing. This can be used for industrial fermentation, so we are consulting specialists and connecting with related industries for advice.

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Reference

Ref. in Background:

[1].Bell JT, Pai AA, Pickrell JK, Gaffney DJ, Pique-Regi R, Degner JF, Gilad Y, Pritchard JK (2011). "DNA methylation patterns associate with genetic and gene expression variation in HapMap cell lines". Genome Biology. 12 (1):
[2]. Austin S, Dixon R (Jun 1992). "The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent". The EMBO Journal. 11(6): 2219–28.
[3].Weaver, Robert J. (2007). "Part V: Post-transcriptional events". Molecular Biology. Boston: McGraw Hill Higher Education..
[4].Hu W, Coller J (2012). "What comes first: translational repression or mRNA degradation? The deepening mystery of microRNA function". Cell Res. 22 (9): 1322–4. doi:10.1038/cr.2012.80
[5].Kozak M (1999). "Initiation of translation in prokaryotes and eukaryotes". Gene. 234 (2): 187–208. doi:10.1016/S0378-1119(99)00210-3. PMID 10395892
[6].Malys N, McCarthy JEG (2010). "Translation initiation: variations in the mechanism can be anticipated". Cellular and Molecular Life Sciences. 68 (6): 991-1003. doi: 10.1007/s00018-010-0588-z.

Ref. in Design:

[1] Xu, C., Huang, R., Teng, L., Jing, X., Hu, J., Cui, G., . . . Xu, J. (2015). Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization. Nat Commun, 6, 6900. doi: 10.1038/ncomms7900.
[2] Carpousis, A. J., Luisi, B. F., & McDowall, K. J. (2009). Endonucleolytic initiation of mRNA decay in Escherichia coli. Progress in molecular biology and translational science, 85, 91-135.
[3]Bricker, A. L., & Belasco, J. G. (1999). Importance of a 5′ Stem-Loop for Longevity ofpapA mRNA in Escherichia coli. Journal of bacteriology, 181(11), 3587-3590.
[4] Zuker M. Mfold web server for nucleic acid folding and hybridization prediction[J]. Nucleic acids research, 2003, 31(13): 3406-3415.
[5] http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form
[6] Yarnell W S, Roberts J W. Mechanism of intrinsic transcription termination and antitermination[J]. Science, 1999, 284(5414): 611-615.