Difference between revisions of "Part:BBa K1051301"
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<h1>Characterization in <i>Cryptococcus neoformans</i></h1> | <h1>Characterization in <i>Cryptococcus neoformans</i></h1> | ||
| − | 1.We used this human condon optimised SpCas9(BBa_K2130013) and sgRNA(BBa_K3506050) in Cryptococcus neoformans. sgRNA was designed to target the ADE2 gene. This gene encoding a phosphoribosylaminoimidazole carboxylase in the biosynthetic pathway of adenine. A loss-of-function mutation in ADE2 results in an adenine auxotroph that forms pink colonies on culture plates that contain a low level of adenine, thereby enabling a visual evaluation of the action of CRISPR-Cas9. Upon transforming the linearized vectors carrying both the Cas9 and the sgDNA cassettes into 4500FOA, a large proportion of URA5-positive transformants formed on the YNBA plates. Then we transferred them to a 4℃ refrigerator. Red colonies were selected and inoculated into YPD medium, then placed it in 30℃ incubator for days, pink colonies grew, indicating that SpCas9(BBa_K2130013) successful targeted at the ADE2 locus in Cryptococcus neoformans. | + | 1.We used this human condon optimised SpCas9(BBa_K2130013) and sgRNA(BBa_K3506050) in Cryptococcus neoformans. sgRNA was designed to |
| + | target the ADE2 gene. This gene encoding a phosphoribosylaminoimidazole carboxylase in the biosynthetic pathway of adenine. A loss-of-function mutation in ADE2 results in an adenine auxotroph that forms pink colonies on culture plates that contain a low level of adenine, thereby enabling a visual evaluation of the action of CRISPR-Cas9. Upon transforming the linearized vectors carrying both the Cas9 and the sgDNA cassettes into 4500FOA, a large proportion of URA5-positive transformants formed on the YNBA plates. Then we transferred them to a 4℃ refrigerator. Red colonies were selected and inoculated into YPD medium, then placed it in 30℃ incubator for days, pink colonies grew, indicating that SpCas9(BBa_K2130013) successful targeted at the ADE2 locus in Cryptococcus neoformans. | ||
[[File: T--XHD-Wuhan-China--literature_figure_1.png |thumb|none|800px]] | [[File: T--XHD-Wuhan-China--literature_figure_1.png |thumb|none|800px]] | ||
<h1>predict the expression speed of Cas9</h1> | <h1>predict the expression speed of Cas9</h1> | ||
Revision as of 11:26, 26 October 2020
clb2 promoter (during G2 phase)
Purpose
Works for the cell synchronization device, B-type cyclin involved in cell cycle progression; activates Cdc28p to promote the transition from G2 to M phase;
Principle
It is a promoter which promotes transciption in G2 of the yeast cell cycle .If you want to express your proteins in G2 of the yeast cell cycle ,you can add the cln2 sequence before your gene sequence ,then put them in a yeast plasmid .After transformation ,you can get the protein you want from the metabolic product of the Bacterial you use for transformation .
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 463
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 437
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
BNU-China 2020 - Contribution
First, we expressed this protein in Cryptococcus neoformans and demonstrated that it can function well. Second, we use a model to predict the expression speed of Cas9. Third, we found an article about Cas9 studied the speed of searching targets.
Characterization in Cryptococcus neoformans
1.We used this human condon optimised SpCas9(BBa_K2130013) and sgRNA(BBa_K3506050) in Cryptococcus neoformans. sgRNA was designed to
target the ADE2 gene. This gene encoding a phosphoribosylaminoimidazole carboxylase in the biosynthetic pathway of adenine. A loss-of-function mutation in ADE2 results in an adenine auxotroph that forms pink colonies on culture plates that contain a low level of adenine, thereby enabling a visual evaluation of the action of CRISPR-Cas9. Upon transforming the linearized vectors carrying both the Cas9 and the sgDNA cassettes into 4500FOA, a large proportion of URA5-positive transformants formed on the YNBA plates. Then we transferred them to a 4℃ refrigerator. Red colonies were selected and inoculated into YPD medium, then placed it in 30℃ incubator for days, pink colonies grew, indicating that SpCas9(BBa_K2130013) successful targeted at the ADE2 locus in Cryptococcus neoformans.
predict the expression speed of Cas9
The speed of Cas9 searching its target
In the paper named Kinetics of dCas9 target search in Escherichia coli, researchers from Uppsala University studied how fast Cas9 can find the target. They used dCas9 to find it out by using single molecule fluorescence microscopy and bulk restriction protection assays. In their study, an artificial chromosome was cloned called pSMART, 36 lac01 sites were on each of it as the search target. To measure the time required for Cas9 to locate by seeing it, they fused the dCas9 to the fluorescent protein YPet and expressed fused protein at a low copy number in a strain containing pSMART plasmids. Without IPEG, lac01 sites were occupied with LacI so that dCas9-YPet can not bind, after adding IPTG, LacI dissociated and dCas9-YPet can bind the sides, then investigators detected the specific fluorescent spots.They considered many aspects that can cause errors to make the result accurately. They obtained the association rate is about 2.7 × 10−3 ± 0.6 × 10−3 min−1 molecule−1 and found that, an individual sgRNA-programmed dCas9-YPet protein requires 6 hours to find and bind its target site on average. After that，they developed a bulk restriction-protection assay to find out the difference of activity between fluorescent fusion protein and native protein, the restriction-protection estimate for the non-fusion dCas9 association rate falls within the range 2.9 × 10−3 ± 1.5 × 10−3 min−1 molecule−1.
Reference
Semlali A , Killer K , Alanazi H , et al. Cigarette smoke condensate increases C. albicans adhesion, growth, biofilm formation, and EAP1, HWP1 and SAP2 gene expression[J]. Bmc Microbiology, 2014, 14(1):1-9.
Introduction of paper2
In this paper，The author has studied HWP1, SAP6 and Rim101 gene expression pattern in C. albicans
Results
The effect of farnesol, CS and AL nanogels containing farnesol on the expression of HWP1, SAP6 and Rim101 genes was investigated using real-time PCR (Figure 7). The finding shows that expression of HWP1 and SAP6 genes in C. albicans treated with 300 mM concentration of farnesol, CS and AL nanogels containing farnesol decreased significantly in comparison with un-treated control group (p<.01). However, it was found the significant difference between the expression of SAP6 gene of C. albicans treated with CS nanogel and non-treated Candida was observed. The expression of Rim101 in Candida treated with CS nanogel significantly decreased as compared to the non-treated cells (p<.01), whereas, Rim101 C. albicans treated with farnesol and AL nanogel containing farnesol did not show any change in the gene expression level.
Reference
Fatemeh Nikoomanesh, et al.Design and synthesis of mucoadhesive nanogel containing farnesol: investigation of the effect on HWP1, SAP6 and Rim101 genes expression of Candida albicans in vitro. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2019, VOL. 47, NO. 1, 64–72
Introduction of paper3
HCR is important for HWP1 expression under multiple growth conditions. HCR (HWP1 control region) is located 1410 bp upstream of the transcription start site of HWP1 gene. HCR is believed to be relevant to the regulation of the gene HWP1. The results showed that when HCR is knocked out, the expression of the gene HWP1 decrease under several growth condition.The reporter strain S is similar to strain -1902GFP except that the HCR region has been deleted.
Results
Reference
Samin K , Bao N Q , Wolyniak M J , et al. Release of transcriptional repression through the HCR promoter region confers uniform expression of HWP1 on surfaces of Candida albicans germ tubes[J]. Plos One, 2018, 13(2):e0192260.
Introduction of paper4
In this paper，The author has studied Hwp1 requirement for biofilm integrity in vitro.
Results
We reported recently that an hwp1/hwp1 mutant produces biofilms in vitro with a slight reduction in biomass compared to the wild- type strain (11). To determine whether this defect is caused by the hwp1 mutation, we compared biofilm biomasses (means standard deviations of results from quadruplicate samples) from the wild-type (0.0089 0.0023 g), mutant (0.0024 0.0002 g), and reconstituted (0.0077 0.0016 g) strains. The hwp1/hwp1 mutant produced a biofilm with threefold less bio- mass than the reconstituted strain (P 0.006). The mutant biomass was also significantly reduced from that of the wild- type strain (P 0.005). The reconstituted strain and wild-type control strain (CAI4-URA3) produced similar levels of biofilm biomasses. These results indicate that Hwp1 is required for normal biofilm formation. Biofilm visualization through CSLM confirmed the biofilm defect of the hwp1/hwp1 mutant (Fig. 1). The mutant produced a biofilm of 100 m in depth that contained few hyphae (Fig. 1A and C). Both hyphae and yeast cells were found in the medium surrounding the biofilm (Fig. 1E). Reconstitution with a wild-type HWP1 allele permitted production of a biofilm of 200 to 300 m in depth in which hyphae were readily apparent (Fig. 1 B and D). Therefore, the mutant defect in biofilm biomass is similar in magnitude to its defect in biofilm depth. The finding that cells are present in the biofilm supernatant suggests that Hwp1 may be required to retain cells within a biofilm.
FIG. 1. In vitro biofilm formation. Biofilms were grown under our standard conditions (13) in Spider medium and stained with concanavalin A for CSLM visualization. Artificially colored CSLM depth views, in which blue color represents cells closest to the silicone and red color represents cells farthest from the silicone, are shown in panels A and B, in which blue represents 0 m and red represents 300 m (panel A) or 500 m (panel B). CSLM side views are shown in lower panels C and D, in which the scale bars represent 50 m. Cells in the surrounding medium of the hwp1/hwp1 biofilm were visualized through phase-contrast microscopy at 400 magnification (panel E)