Difference between revisions of "Part:BBa K1051900"

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<p>Finally we set down that kass1 = kdis1.
 
<p>Finally we set down that kass1 = kdis1.
 
</p>
 
</p>
<img src="https://static.igem.org/mediawiki/igem.org/0/01/Kass1_eq_kdis1.png">
+
https://static.igem.org/mediawiki/igem.org/0/01/Kass1_eq_kdis1.png
 
<center><p>Figure. Simulation Result of Two mRNA when Kass1=Kdis1
 
<center><p>Figure. Simulation Result of Two mRNA when Kass1=Kdis1
 
</p></center>
 
</p></center>
Line 130: Line 130:
 
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p>
 
<p>As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.</p>
 
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho &amp; Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p>
 
<p>The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho &amp; Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.</p>
<img src="http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg" title="Principle of Sic1" description="Over expression of Sic1 can stop all yeast cells to G1 phase.">
+
http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg
<p>Figure. Over expression of Sic1 can stop all yeast cells to G1 phase.</p>
+
<center><p>Figure. Over expression of Sic1 can stop all yeast cells to G1 phase.</p></center>
  
 
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p>
 
<p>After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).</p>
<img src="https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png" title="Phosphorylation Sites" description="N-terminal phosphorylation sites to be mutated.">
+
https://static.igem.org/mediawiki/parts/8/8b/Sic1-mutate.png
<p>Figure. N-terminal phosphorylation sites to be mutated.</p>
+
<center><p>Figure. N-terminal phosphorylation sites to be mutated.</p></center>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 01:21, 3 October 2013

G1 phase magic with cell synchronization device and CRISPRi induced alternative splicing device.

clb5 +d-box +GFP + src1 intron + tom40 + cca + gal + dCas9 + cca + snr52 + sgRNA(Hub1) + sup4

Alternative Splicing Device

In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.

Src1 Intron and Hub1

SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.

Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions.

Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older.

Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.

SICI_principle.jpg

Figure. Principle for alternative splicing of Src1.

Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.

dCas9 CRISPR interface system

CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.

Figure4._The_structure_of_sgRNA.png

Figure. sgRNA structure.

CRISPRi_principle.png

Figure. Principle of the function of dCAS9 and guide RNA.

Alternative Splicing by CRISPRi

To predict the alternative outcome, we also made an intron model to show different results due to incubating in different media. In our project, intron can be spliced in two different ways, providing a completely different outcome because of frame-shift, and this result is not a change like 1-0 to 0-1, but somehow more like a change between 0.4-0.6 and 0.8-0.2.

DCas9.png

Figure. dCas9 Controlled SRC1 Intron Splicing

Reactions:

Formula1.png Formula2.png

Parameter Table:

Parameter

Explanation

P(dCas9_m)

dCas9 mRNA transcription rate

P(sgRNA)

sgRNA transcription rate

P(dCas9p)

dCas9 protein translation rate

D(RNA)

Average degradation rate of RNA

Kass

Association rate of CRISPRi system

Kass

Association rate of CRISPRi system

Kdis

Dissociation rate of CRISPRi system

Kass1

Association rate of modified spliceosome

Kdis1

Dissociation rate of modified spliceosome

Kdis1

Dissociation rate of modified spliceosome

K

Splicing rate

P(Hub1_m)

Hub1 mRNA transcription rate

P(Hub1_P)

Hub1 protein translation rate

P(pre-mRNA)

pre-mRNA transcription rate

P(ProteinL)

5’L protein translation rate

P(ProteinS)

5’S protein translation rate

D(Protein)

Average degradation rate of protein

There are 4 parameters that we cannot find during our research, including kass, kdis representing the association and dissociation rate of CRISPRi system, and kass1 and kdis1 representing the association and dissociation rate of Hub1p and spliceosome.

We run parameter scan for each system individually, and found out that with CRISPRi system is more efficient with higher kass and lower kdis, as expected.

And about the alternative splicing model, we attempted to fit simulation result to experimental one. In Hub1 expressed system, L-mRNA will rise at first but descend to an equilibrium stage while S-mRNA will directly rise to its own equilibrium stage.

kass1 should be larger than kdis1, or L-mRNA will be produced more than S-mRNA instead of a ratio of 40-60.

Kass1_lt_kdis1.png

Figure. Simulation Result of Two mRNA when Kass1<Kdis1

Also, kass1 should not be too smaller than kdis1, or their ratio will be much larger than 40-60.

Kass1_gt_kdis1.png

Figure. Simulation Result of Two mRNA when Kass1>Kdis1

Finally we set down that kass1 = kdis1.

Kass1_eq_kdis1.png

Figure. Simulation Result of Two mRNA when Kass1=Kdis1

When inhibiting the expression of HUB1, there is still a background splicing of 5’S site, so we need another parameter b in S-splicing.

Background S-splicing parameter b is mostly related to the ratio of spliceosome and Hub1p-modified spliceosome (Hub1_spliceosome) at equilibrium state. With higher b, L-mRNA and S-mRNA will come closer at equilibrium stat while it influence no-Hub1p situation more than Hub1p situation.

Parameter_scan_of_b_%28gal%29.png

Figure. Parameter Scan of b with Galactose Input

Parameter_scan_of_b_%28no_gal%29.png

Figure. Parameter Scan of b without Galactose Input

Cell Synchronization

As we known, the yeast cell cycle contains a huge and complex regulatory network in the transcription level, translation level as well as protein level. In our project we utilize Sic1 as a regulator to help elongate G1 phase in yeast cell.

The activation of B-type cyclin (Clb5/6)+Cdc28 kinases is a necessary step for initiation of DNA replication in vivo. One of its inhibitor Sic1 can be phosphorylated by the activated Cln+Cdc28, thereby targeted for degradation. Over expression of the gene encoding p40, SIC1, produces cells with an elongated bud morphology(figure1)(Nugroho & Mendenhall, 1994). SIC1 deletion is viable and causes increasing frequencies of chromosomes broken and lost. The deletion strain segregates out many dead cells, which are primarily arrested at the G2 checkpoint in an asymmetric fashion. Therefore, it has an important role in ensuring genomic integrity, and that this role has a pronounced mother-daughter asymmetry.

http://upload.wikimedia.org/wikipedia/commons/0/06/Sic1_David_Morgan10-5.jpg

Figure. Over expression of Sic1 can stop all yeast cells to G1 phase.

After phosphorylation, phospho-Sic1 is specifically recognized by the F-box protein Cdc4, which leads Sic1 being ubiquitinated by the Cdc34±SCF complex (E3). The recognizing and binding by Cdc4 is based on the Sic1’s 9 Cdc4 phospo-degrons (CPDs, figure. a). Several phosphorylation sites contributed to Sic1 instability, with an order of Thr45, Ser76, Thr5,Thr33 and other less significant sites. The immunocipients after selectively phosphorylation as figure e showed suggested that at least six sites phosphorylation is necessary for the Cdc4 recognizing and binding with Sic1. Furthermore, the culture of GAL1-SIC1 constructs strain showed less than 6 sites are phosphorylated is not sufficient for SIC1 degradation in vitro(Nash et al., 2001).

Sic1-mutate.png

Figure. N-terminal phosphorylation sites to be mutated.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 6680
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 5088
    Illegal AgeI site found at 1943
    Illegal AgeI site found at 7092
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5234
    Illegal BsaI site found at 6088
    Illegal BsaI.rc site found at 1396
    Illegal BsaI.rc site found at 3759
    Illegal SapI site found at 1745
    Illegal SapI.rc site found at 7807



References

[1]Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol.
[2]Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., . . . Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602-607.
[3]Mishra, S. K., Ammon, T., Popowicz, G. M., Krajewski, M., Nagel, R. J., Ares, M., Jr., . . . Jentsch, S. (2011). Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing. Nature, 474(7350), 173-178.