Team:BM-AMU- 2019.igem.org

Experiments

Plasmid Construction

In order to construct a dual fluorescence reporter cell model, we constructed pBKS-hECad-tdTomato （BBa_K3120018）and pBKS-hNCad-EGFP vector plasmids （BBa_K3120017）. Next, we cloned selected sgRNAs into Px330 vector to knock the fluorescent protein into the target gene by CRISPR/cas9 technology. These plasmids are detailed in Figure 1.

Figure1

pBKS-hECad-tdTomato（BBa_K3120018） : composing of pBluescriptIclI SK vector skeleton, the left segment of termination codon of E-Cadherin CDS sequence is homologous left arm, and the right segment of termination codon is homologous right arm, and the tdTomato CDS sequence inserted in the middle.

pBKS-hNCad-eGFP（BBa_K3120017） : composing of pBluescriptIclI SK vector skeleton, the left segment of termination codon of N-Cadherin CDS sequence is homologous left arm, and the right segment of termination codon is homologous right arm, as well as the inserted eGFP CDS sequence and loxP-Neo-loxP. Neo resistance genes play a role in the absence of Cre enzyme. Because N-Cadherin does not express in the epithelium, it is impossible to identify the insertion of eGFP after N-Cadherin by fluorescence, and then to screen the Neo resistance genes. After successful screening, Gre enzyme was added and loxP-Neo-loxP was removed.

pX330（BBa_K3120015, BBa_K3120016）: composing of U6 promoter, 20 bp long gRNA, CAS9 and AMP resistance genes. The cas9 protein first binds to gRNA, then forms a complex structure with DNA under the guidance of PAM sequence, and then cuts the double strands of target DNA.

In order to manipulate the concentration of exogenous factor Dox/Tamoxifen to artificially regulate the expression of EMT-TFs (Snail1, Twist1) in cells and to obtain EMT cells in different states, we constructed LC-TetO-IN-hSnail1, pPyIZ-hTwist1-ERT2, pPyIZ-tTR-KRAB plasmids.These plasmids are detailed in Figure 2.

Figure2

pPyIZ-TetR-KRAB（BBa_K3120013）: After CAG promoter, TetR and KRAB coding sequences were connected in series, including a nuclear localization signal NLS, and IRES introduced Zeocin resistance gene.

pPyIZ-hTwist1-ERT2 （BBa_K3120020）: The TWIST1 CDS region was linked to ERT2 coding sequence by CAG promoter. IRES was introduced into Zeocin resistance gene.

LC-TetO-IN-hSnail1（BBa_K3120019）:Seven tandem TRE sequences and CAG promoters constitute TetON promoters. SNAIL1 CDS and IRES in tandem introduces NEO resistance genes. LTR sequences are introduced at the 3'and 5'ends of this part, respectively.

Construction of Monitor

We designed a knock-in strategy for a reporter cassette that results in a gene fusion facilitating the concordant expression of E-Cadherin and td-Tomato. Briefly, 90% confluent hES cells WA09 (WiCell) were digested into single cells with accutase. Cells were centrifuged and resuspended with the CRISPR construct and reporter plasmid in nucleofection media (Lonza). Cells were electroporated and then cultured on plates in mTESR1 media with ROCK inhibitor. After 48 h, cells were digested and single cell was inoculated into the 96 well plate by FACS sorting. After transfecting the plasmid pBKS-hE-Cadherin-tdTomato and PX330 into WA09 cells for CRISPR/cas9 technology to knock in genes, we selected more than four clones to identify whether the fluorescent protein tdTomato was inserted into the correct site at the genome level. We proved the single-cell clone E1,E2 and E4 was correct, which fluorescent proteins tdTomato knocked into a piece of DNA in the genome that fits our goals.While the E3 is wrong. Next, we validated clone E1（WA09 E1） at genome and cDNA levels, respectively.The result shows that the fluorescent protein tdTomato was inserted at the correct location. To investigate whether the fusion expression of fluorescent protein tdTomato and E-Cadherin affected the degradation of the protein, Actinomycin treatment (18.75umol/mL, The duration of drug treatment is 0hr,3hr,6hr,12hr and 24hr ) was performed. The results of Western blot showed that protein degradation efficiency of fusion expressed protein was consistent with that of E-Cadherin. We also observed the subcellular localization of the cells, and the results showed no difference from those reported in the literature.The above results indicate that tdTomato inserts into the correct site and function normally.

In order to establish the fluorescence model, we need to transfer pBKS-hECad-tdTomato （BBa_K3120018） and pBKS-hN-Cad-eGFP （BBa_K3120017） into cells to function. Lipofectamine 2000 Reagent and Lonza electroporation were used. Fluorescence microscopy showed that our transfection was successful. Next, in order to confirm that the fluorescent protein did knock into the correct site, we selected the cells for monoclonal and flow sorting, extracted RNA, PCR analysis and Western Blot from the selected cells. The results showed that the fluorescent protein and target gene were successfully fused and expressed.

Construction of Controller

Transfected plasmids pPyIZ-hTwist1-ERT2 （BBa_K3120020） into cells WA09 E1 by Lipofectamine 2000 Reagent.The transfected cells were screened with 10μg/ml zeocin. In order to obtain different states of EMT cells by changing the concentration of Tamoxifen，We set up the concentration gradient and time gradient of Tamoxifen respectively, and detected the mRNA expression of EMT-related genes by qPCR.

In order to obtain a cell line which has stable Tet-on system, lentivirus was used for transfection. LC-TetO-IN-hSnail1 （BBa_K3120019） was constructed and co-transfected with packaging plasmids pSPAX2 and pMD2G respectively. The plasmids were transfected into 293FT cells according to the instructions of Lipofectamine 2000. When obvious cytopathic changes occur (transfection 72 hours), culture supernatant was taken and virus was taken by centrifugation at 6000 r/min.Infecting cells with the resulting lentivirus （MOI=5）.10 μg/ml zeocin was used to screen the transfected cells. Then we transfected pPyIZ-TetR-KRAB( （BBa_K3120013） ). In order to obtain EMT cells in different states by changing DOX treatment，We set up the concentration gradient and time gradient respectively, and detected the mRNA expression of EMT-related genes by qPCR.

Experiments for Bronze and Gold medals

In the construction of controller, CAG Promoter is used in Tet-on and Tamoxifen induced expression system, but up to now there is no quantitative data table. In order to know the strength of CAG promoter, we designed experiments to compare the efficiency of CAG promoter with other common promoters.Detailed design: We constructed four plasmids. In the pGuide vector skeleton, CAG, CMV, EF1a and Ubiquitin promoters were used to initiate the over-expression of GFP, and the expression intensity of GFP represented the strength of promoter. After four plasmids were constructed, these plasmids were transfected into HEK293FT cells by Lipofectamine 2000 Reagent, and GFP was successfully expressed. Samples were collected 24 and 48 hours after transfection. Flow cytometry was used to compare the expression of GFP, and finally the strength of these promoters was obtained.

In addition, due to the leakage problem of the classical Tet-on system, we improved the classical Tet-on system and fused TetR with KRAB. The suppressed gene is expressed when Dox is added. In order to prove that our modification is effective, we compared efficiency of rtTA, TetR and TetR-KRAB. The specific design is: Improvement Verification Experiment of TetR-KRAB. Equal amount of HEK293FT cells were seeded in a 6-well plate ，3 groups co-transfected with plasmids LC-TetO-GFP and plasmid expressing repressors severally. Flow cytometry was used to detect GFP expression on 5, 7, 9 days after transfection. On the day 9, DOX (1 μg/μL) was added to initiate downstream gene expression, and GFP expression was detected 5 days later. HEK293FT cells were used as blank control at each stage.

FACS-Seq Sample Preparation

After the successful construction of Monitor and Controller, we obtained EMT cells in different intermediate states by changing the dose and time of Dox/Tamoxifen treatment. Next, cells were sorted into several populations according to their fluorescence intensity. The transcriptome data of each sample was then analyzed using RNA-seq.

Transcriptome sequencing

The experimental process:

1) Sample of RNA by oligo T primers into reverse transcription reaction, generating the first cDNA chain;

2) Generate double-stranded cDNA through the joint reaction of RNase H, DNA polymerase and T4 ligase;

3) Double chain cDNA by Tn5 enzyme fragmentation, and cover the RD sequencing on the double end sequence below;

4) Through (3) combined with RD sequences at the ends of the connection attributes (P5, se-quencing primers on both ends of the P7, hand in hand enrichment of PCR amplification;

5) Library mixed for sequencing.

The flow chart of Data analysis

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Team:BM-AMU/Experiments