Part:BBa_K1051900

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

clb6 +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.

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.

Figure. sgRNA structure.

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.

Figure. dCas9 Controlled SRC1 Intron Splicing

Reactions:

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.

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.

Figure. Simulation Result of Two mRNA when Kass1>Kdis1

Finally we set down that kass1 = kdis1.

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.

Figure. Parameter Scan of b with Galactose Input

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).

Figure. N-terminal phosphorylation sites to be mutated.

Targeting Peptides

A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in cell, including nucleus, mitochondria, endoplasmic reticulums (ERs), chloroplasts, apoplasts, peroxisomes and plasma membrane. Targeting peptide can exists in both N-terminal, C-terminal and internal sequence of a precursor protein. And after transported, some target peptides are cleaved by signal peptidases.

In our project we utilized 19 peptides target to 9 sub-locations in yeast cells, and when combined with fluorescent proteins, such region can be marked by different colors.

Mitochondria

Though it accounts a small ratio in the cell space, mitochondria possess about 10% to 15% proteins encoded by nuclear genes in eukaryotic organisms. These proteins are synthesized in cytosol and then recognized by the membrane receptors of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intra-mitochondrial sorting of these proteins. ATP is used as an energy source; Chaperones and auxiliary factors assist in folding and assembly of mitochondrial proteins into their native, three-dimensional structures.

Figure. Mitochrondria import pathway.

As shown in the figure above, beta-barrel outer-membrane proteins (dark green), precursor proteins (brown) with positively charged amino-terminal presequences and multispanning inner-membrane proteins (blue) with internal targeting signals are recognized by specific receptors of the outer mitochondrial membrane (TOM) translocases Tom20, Tom22 and/or Tom70. The precursor proteins are then translocated through a small Tom proteins of the TOM complex, Tom40 pore, which the TOM complex contains two or three.

Promoter Verification

Achievements

Using the GFP as reporter and morphological alteration as cell cycle representation, we verified the Clb6 can be activated in G1 phase in the yeast.

As shown in the picture in normal lights, there are some yeast were budding. These budded yeast cells and small size budding yeasts (within the red circles) are assumed in the G1 phase of cell cycle. And the next picture was taken under activation light, thus the green lights can representing the Clb6 promoter expressing. The two pictures indicated the Clb6 were expressed in the G1 phase as we expected when designing the experiments

PS: we modified the contrast ratio to lower the lights of neighbor cells, thus our results looking better.

Targeting Peptides

The four pictures shows the reporters can rightly locate to Mitochondria, Nucleus and Vacuole，respectively. Picture A is the negative control, all yeast cells are lighted with GFP. And figure B is the reporter to the mitochondria, we can saw there are several light spots in one cells. Figure C is the reporter located in nucleus, the green spots are small and there is only one in a yeast cell.The last picture shows the reporter of Vacuolar membrane, the green lights were discrete in cells which was as expected.

Figure. Test for targeting peptides to mitochondria, nucleus and vacuolar.

Microfluidics Device

We made the chip as a platform for watching and synchronize the cells. First step is capture the cells by the chip. As showed in the two figures, both E.coli and budding yeast can be captured by the chip successfully.

Figure. E. coli and yeast cells have been captured by the chip successfully.

As Sic1 method has not been successful, we tried another way by changing medium by microfluidics. Protocols can be found in Notes page.

Figure. All cells are synchronized to G1 phase by using the microfluidics method.

All yeast cells in microfluidics chip are in G1 phase, after budding.

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.

Characterization by 2021iGEM_Xiamen_City

Improvement of an existing part

Compared to the old part BBa_K1051900, G1 phase magic with cell synchronization device and CRISPRi induced alternative splicing device, we design a new part BBa_K4002007, which contains the HCas9 protein. The HCas9 protein is a human optimized Streptococcus pyogenes Cas9. And its N and C terminal are inserted with SV40 NLS peptide.

The group iGEM13_Shenzhen_BGIC_ATCG wish to grasp the usage of cell cycle tools, and they wanted to direct refined actions in a cell by CRISPR Cas9 technology.

Our team design the new composite part BBa_K4002007 to express HCas9, with sequence different from the old one BBa_K1051900 (Figure 7). We thought of using CRISPR-Cas9 technology to achieve heterologous expression of endo-pgaA, an endo-galacturonase gene from Aspergillus niger SC323, in fruit wine yeast to obtain a strain that can both degrade pectin and ferment alcohol.

Figure 7. The blast results about the sequence of our new part BBa_K4002007 and the old one BBa_K1051900.

Our project will help explore the development of multifunctional fruit wine yeast, reduce the production cost of fruit juice and fruit wine, and contribute to the development of food industry production and modern brewing engineering.

Cas9+gRNA+HR-L-endo-pgaA-HR-R

Usage and Biology

Saccharomyces cerevisiae is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. In fruit wine production, Saccharomyces cerevisiae uses the sugars in fruit juice to ferment to produce alcohol. It is necessary to add pectinase to destroy the pectin in the cell wall to increase the juice yield and increase the dissolution of aromatic substances such as pigments or terpenes. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are specific regions in some bacterial and archaeal genomes that, together with associated Cas (CRISPR-associated) genes, function as an adaptive immune system in prokaryotes. While the specific ‘adaptive’ nature of this immunity is still under investigation, it is known that exogenous DNA is processed by Cas proteins into short (~30 base pair) sequences that are adjacent to the Protospacer Adjacent Motif (PAM) site. These short pieces of DNA are then incorporated into the host genome between repeat sequences to form spacer elements. The repeat-spacer-repeat array is constitutively expressed (pre-CRISPR RNAs or pre-crRNAs) and processed by Cas proteins to form small RNAs (crRNAs). The small RNAs are then loaded into Cas proteins and act to guide them to initiate the sequence-specific cleavage of the target sequence.

Figure 1. The principle of CRISPR Cas9.

Construct design

pHCas9 is a sequence coding endonuclease enzyme associated with the CRISPR. sgRNA is a sequence of RNA participant in CRISPR. HR-L is the homology arm upstream HXK1. HR-R is the homology arm downstream HXK1. pgaA is the sequence of pgaA inserted in the homology arm (Figure 2).

Figure 2. Cas9+gRNA+HR-L-endo-pgaA-HR-R box.

The profiles of every basic part are as follows:

BBa_K4002000

Usage and Biology

This is a coding sequence of left homology arm, refers to the flanking sequence on one side of the HXK1 sequence, which is completely consistent with the genome sequence, and is used to identify and recombine the region. system (T6SS).

BBa_K4002001

Usage and Biology

This is a coding sequence of right homology arm, refers to the flanking sequence on one side of the HXK1 sequence, which is completely consistent with the genome sequence, and is used to identify and recombine the region.

BBa_K4002005

Usage and Biology

Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and poly-alpha-1,4-galacturonide glycanohydrolase.

BBa_K4002004

Usage and Biology

This is a sequence coding pHcas9 protein. This protein is a dual RNA-guided DNA endonuclease enzyme associated with the (CRISPR) adaptive immune system. The Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. The genes that encode the Cas9 protein and sgRNA were introduced into a cell and programmed to change its target gene. sgRNA has regions that are complementary to the target sequence. A complex consisting of sgRNA and Cas9 protein is formed inside the cell and binds to target sites.

BBa_K4002003

Usage and Biology

BBa_K4002003 is a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they form complexes with. And this sequence is inserted into plasmid vector.

Experimental approach

1. Construction of CRISPR expression plasmids The PgaA is an enzyme that hydrolyzes the α-1,4 glycosidic bonds between galacturonic acid residues present in polygalacturonan in plant cell walls and therefore facilitate plant cell wall breakdown. In the production of fruit wine, pectinase has been used to destroy the pectin in the cell wall in order to improve the juice yield and increase the dissolution of aromatic substances such as pigments or terpenes.

Figure 3. Construction CRISPR expression plasmids. (A) Schematic representation of CRISPR expression vectors; (B) Agarose gel electrophoresis of pHCas9-Nours (lanes 1 and 2) and pYES2–gRNA-hyg-MCS (lanes 3 and 4) plasmids.

We sought to integrate PgaA gene into S. cerevisiae genome based on the CRISPR technology in order to obtain yeast strains that not only produces alcohol but can also decompose pectin. To this end, we designed two plasmids expressing Cas9 and gRNA (Fig. 3A), as well as the repair template. The agarose gel electrophoresis results indicated that the plasmids of pHCas9-Nours and pYES2–gRNA-hyg-MCS were extracted from DH5 bacterial cells with high quality and could be used for following transformation experiments.

2. Construction of repair template The repair template DNA containing PgaA gene (Fig. 4A) was generated by the overlap-PCR method. Firstly, the DNA fragments of upstream and downstream homologous regions were amplified with ~20 bp ends overlapping to the PgaA gene, producing ~500 bp PCR products (Fig. 4B). Secondly, the two fragments were annealed to the 5’- and 3’-ends of PgaA. Finally, the annealed products were further amplified using end primers of HR-L and HR-R, which resulted in a fragment of 1.5 kbas verified by agarose gel electrophoresis and DNA sequencing (Figs. 4C and 4D).

Figure 4. Construction of repair template. (A) Schematic representation of repair template; (B) Agarose gel electrophoresis of PCR products; (C) DNA sequencing result analysis.

3. Yeast strain transformation and positive transformants verification The constructed CRISPR plasmids and repair template DNA were chemically transformed into the S. cerevisiae strains. The positive transformants were selected against YPD medium supplemented with Nours and hygromycin. The resulting colonies were picked up and cultured. To investigate whether the PgaA gene was integrated into yeast genome, we performed PCR experiments using the upstream and downstream primers complementary to HR-L and HR-R genes, respectively. As shown in Fig. 5A, we obtained specific PCR products with expected size of ~1500 bp. The DNA fragments were then extracted and purified for sequencing. The sequencing results finally confirmed that the PgaA gene was successfully integrated into S. cerevisiae genome (Fig. 5B).

Figure 5. Verification of PgaA containing transformants. (A) Agarose gel electrophoresis of PCR products; (B) DNA sequencing result analysis.

Proof of function

Pectinase activity assay The pectinase activities of PgaA were determined using the dinitrosalicylic acid (DNS) colorimetric method. Briefly, in the presence of PgaA, pectin can be degraded into galacturonic acids, which reacts with DNS to form a compound with a maximum absorption at 540 nm. Thus, the activity of PgaA can be calculated by measuring the absorbance of the reactants with a spectrophotometer. For accurate quantification, a standard curve was generated using a series of concentrations of pectinase standards. As shown in Table. 1 and Fig. 6, the concentration of enzyme correlates well with the absorbance detected at 540 nm, applying to the Lambert-Beer law.

Table 1. Measurement of standard pectinase activities at different concentrations.

Figure 6. Standard curve of pectinase.

With this standard curve, we next determined the concentration of PgaA from recombinant S. cerevisiae strains. Samples from the culture media, total cell lysates and the soluble portion of cell lysates were collected and subjected to DNS colorimetric assay. As shown in Table. 2, the concentration of PgaA in the culture media of sample -1 and -2 were determined at about 0.034 mg/ml and 0.028 mg/ml, respectively, which were relatively higher than that of cell lysates (0.009 mg/ml and 0.007 mg/ml), suggesting that most of the PgaA proteins were secreted into the culture media. In addition, in the cell lysates of sample 1, we detected ~76% of PgaA in the soluble supernatants, implying that most of the PgaA in cells are soluble.

Table 2. Measurement of PgaA concentration and unit of activity in various samples.

We successfully prepared genetically engineered wine yeast strain which contains pectinase in its genome. The pectinase produced from yeast well degrade pectin into small sugars.

References

1.Nishimasu, H., et al. Cell. 2014

2.James E.D., et al. Nucleic Acids Res. 2013

3.https://www.asme.org/topics-resources/content/8-ways-crisprcas9-can-change-world

4.https://www.nature.com/articles/s41599-019-0319-5

5.https://www.sciencedirect.com/topics/food-science/fruit-wine

6.崔凯宇,李迎秋.果胶酶生产和应用的研究进展[J].江苏调味副食品,2016(01):11-13.

7.Yang J , Luo H , Jiang L , et al. Cloning, expression and characterization of an acidic endo-polygalacturonase from Bispora sp. MEY-1 and its potential application in juice clarification[J]. Process Biochemistry, 2011, 46(1):272-277.

8.李烨青. 真菌来源的嗜热果胶酶基因挖掘及其催化效率的改造[D].江西农业大学,2017.

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