iGEM 2019 — Team Shanghai-United

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# Team:Shanghai-United/Notebook

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The characterization protein and early diagnosis of cervical cancer

Here we demonstrate specific protein expression variations related to NFX1 over-expression, a gene found to be closely associated with cervical cancer.

### NoteBook

iGEM Lab Summary

Day 1 – Preparation of experiment equipment and safety training
The first day of experiment we learnt the safety requirements for doing chemistry lab.

General precautions training we learnt to never eat or drink in the lab, keep the work areas clean all the times; keep all combustive material away from open flames; never put our face near the mouth of a chemical container; wear lab clothes - this is our favorite part, we have to war the white doctor like long working garment that make us looking really professional and cool!

Handling chemicals training we learnt to double-check labels; avoid contamination; keep container away from our body; avoid touching chemicals with our hands; wear latex gloves – that green latex gloves is a must for us, and we felt safer with them on.

Handling glassware training we learnt to carry glass tube vertically; never put hot glassware directly on a table; never touch broken glass with our bare hands.

Heating substances training we learnt to use extreme caution with gas burners; never heat anything unless instructed; never heat a closed container.

We were very glad to have use 1 day to do the safety training, cause we never had any safety accidents throughout the whole experiments thanks to our professors' patience and insistence on the safety training.

Day 2 - PCR experiment

The 2nd day Wet Team 9 students were brought to Shanghai Institute of Pharmaceutical Research Lab. Dr. Lu briefly introduced the experiment background and restated safety requirements, particularly some basic experiment operations. They learned the main technics on how to use many lab instruments such as liquid moving gun and 0.2ml EP pipe.

ThepurposeofPCRis to amplify small amounts of a DNA sequence of interest so it can be analyzed separately. PCR can be used to make a large amount of a specific piece of DNA or to test a DNA sample for that sequence. PCR is used for research when it is necessary to make a large amount of a single gene, such as for genetic engineering or cloning.

In the diagnosis of AIDS, PCR can be used to detect the small percentage of cells infected with HIV-1. Following amplification and gel electrophoresis, the presence of an appropriate sized PCR product indicates the presence of HIV-1 sequence and therefore, HIV infection. Summary: PCR is a powerful biochemical technique that enables.

Human papillomavirus(HPV)is a group of more than 150 related viruses. Some types of HPV are considered high risk because they can cause cancer. HPV testingdetects the genetic material (DNA or messenger RNA) of high-risk HPV (hrHPV), primarily to screen for cervical cancer or to determine whether you may be at risk of cervical cancer.

We studied Green Fluorescent Protein, GFP. And did GFP PCR. It was a technique that requires great care to complete. But it was so powerful because you can take very small amounts of DNA and make tons of it.

We did many times of PCR. The number of strands of DNA after each cycle of PCR steps doubled, so the amount of DNA produced was exponential. In this way, we created billions of strands of the GFP DNA. PCR draw out the GFP and then we would go through DNA Agar sugar gel electrophoresis to verify the PCR results. This way to make sure the accuracy of PCR's purpose strips. Then we can cut corner spare.

In order to do the electrophoresis experiment, we need prepare the agorose gel.

While we were waiting for the solution to cool, Dr. Lu taught us how to design Primers. The Primer is a very common Molecule cloning software tool. We learned it so that we can be fully prepared for later on experiments. Dr. Lu taught us in detail about the primer design and experiment arrangement that used in the inoculation into Enzyme cut-carrier of NFX1 and GFP.

 It is crucial to understand how to successfully design primers. Understand the Basic Rules. The primer you design impacts the entire DNA amplification process. DNA polymerases, the enzymes that catalyst DNA replication, can only initiate the replication process by adding nucleotides to primers.

Day 3 - EGFP Gene Editing

Genome editing is a technique used to precisely and efficiently modify DNA within a cell. It involves making cuts at specific DNA sequences with enzymes called ‘engineered nucleases’. Genome editing can be used to add, remove, or alter DNA in the genome.

In this experiment, we need to use CRISPR/Cas9 technology toenable targeted gene editing; we need learn how to Split EGF. to study protein-protein interactions. In this case, two portions of EGFP are fused to the proteins of interest, and when they come into close proximity, the two halves of EGFP undergo folding, maturation, and fluorescence. Since this gen editing is widely used in HPV cervical cancer gene research. To practice the gene editing and get familiar with all the procedures are very crucial for advanced HPV cervical cancer gene research.

Procedures:

1. Recovery of GFP target gene in the DNA gel
2. Double enzyme cut GFP target gene, nurture sticky end
3. Electrophoresis verification on enzyme extraction
4. Recovery of enzyme extraction to DNA gel
5. Determine the concentration of gel extraction
6. Link the carrier and target gene GFP base on Mole ratio 1:10.

Below are practical applications that use GFP Genome editing.

1. Using a new tool for editing genomes, known as CRISPR, researchers have genetically engineered immune cells and improved the ability of these cells to kill cancer cells in mice.

[<https://www.cancer.gov/news-events/cancer-currents-blog/2017/crispr-immunotherapy>]

1. Editing HPV's Genes To Kill Cervical Cancer Cells. Duke University researchers were able to selectively destroy two viral genes responsible for the growth and survival of cervical carcinoma cells, causing the cancer cells to self-destruct. The findings could pave the way toward antiviral strategies targeted against other DNA-based viruses like hepatitis B and herpes simplex. Because this approach is only going after viral genes, there should be no off-target effects on normal cells.

[<http://www.sciencedaily.com/releases/2014/08/140808163449.htm>]

1. One gene editing trial involves the first-ever attempt to use CRISPR to edit cells while they are inside the body. The aim is to prevent cervical cancers by targeting and destroying the genes of the human papillomavirus (HPV) that cause tumour growth. This study is at the First Affiliated Hospital of Sun Yat-Sen University in China.

[<https://www.newscientist.com/article/2133095-boom-in-human-gene-editing-as-20-crispr-trials-gear-up/#ixzz5zTsoY1vs>]

Day 3 - NFX1-123 Gene Editing
The high-risk human papillomavirus (HR HPV) E6 oncoprotein binds host cell proteins to dysregulate multiple regulatory pathways, including apoptosis and senescence. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and together they post-transcriptionally increase hTERT expression, the catalytic subunit of telomerase.
[<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838236/>]

Because the close relationship of NFX1-123 cellular protein with HPV16 E6, our experiment targeted to understand more on how they interacts and results of the interactions. From the experiments, we may have more clues for their relationship, and our experiments can contribute more on HPV study.

Procedures:

1. Draw out the NFX1 gene using PCR method.
2. Verify the PCR gel extraction through Agar sugar gel electrophoresis.
3. Put the gel into the refrigerator for tomorrow’s use.

Day 4 - EGFP Gene Editing

The Escherichia coli strain DH5α is used extensively in recombinant DNA technology. E. coli is a derivative of the popular DH5α. It is T1 phage resistant and endA deficient for high-quality plasmid preparations. Its high efficiency strain is ideal for a wide variety of applications. So our experiment use and get familiar with the Escherichia coli DH5a characteristics and our success of experiment is easier to reach.

Procedures:

1. Inculcate the linked output (of Day 3) into Escherichia coli DH5a
2. Recovery Escherichia coli DH5a, spread evenly after centrifugal onto Solid medium (Ampicillin resistance)

Day 4 - NFX1-123 Gene Editing

Today’s NFX1-123 we did real gene editing (enzyme cut and link the carrier and target gene NFX1) based on Day 3 prepared NFX1 DNA gel. This procedure is exactly same as the procedure we did on Day 3 of EGFP Gene Editing.

Procedure

1. Recovery of NFX1 target gene in DNA gel
2. Double enzyme cut NFX1 target gene, nurture sticky end
3. Electrophoresis verification on enzyme extraction
4. Recovery of enzyme extraction to DNA gel
5. Determine the concentration of gel extraction
6. Link the carrier and target gene NFX1 base on Mole ratio 1:10.

A significant contributor to women's cancer mortality worldwide is cervical cancer, which is caused by high-risk human papillomavirus (HR HPV). While NFX1-123 had been found has fundamentally correlation with HPV E6. Below are practical applications that use NFX1-123 Genome editing on HPV research.

1. Human papillomavirus (HPV) is the most prevalent sexually transmitted infection, affecting an estimated 11% of the world’s population. The high-risk HPV types (HR HPV) account for approximately 5% of the global burden of cancer and thus cause high morbidity and mortality. Although it is known that persistent infection with HR HPV is the greatest risk factor for developing HPV-associated cancer, and that the HPV early proteins E6 and E7 dysregulate immune detection by its host cells, the mechanisms of immune evasion by HR HPV are not well understood. Previous work in the laboratory identified the endogenous cytoplasmic host protein NFX1-123 as a binding partner of the HR HPV type 16 oncoprotein E6 (16E6). Together NFX1-123 and 16E6 affect cellular growth, differentiation, and immortalization genes and pathways.

[<https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187514>]

1. NFX1-123 is highly expressed in cervical cancer and increases growth and telomerase activity in HPV 16E6 expressing cells.e now report that NFX1-123 is highly expressed in primary cervical cancers. In vitro, cells expressing 16E6 and over-expressing NFX1-123 have extended active growth, decreased senescence marker staining, and more rapid cell cycling compared to 16E6 expressing cells with endogenous amounts of NFX1-123.  In cells that express HR HPV E6, greater expression of NFX1-123 can modify active cellular growth and augment hTERT expression and telomerase activity over time, potentially supporting the initiation and progression of HPV-associated cancers.

       [<https://www.ncbi.nlm.nih.gov/pubmed/30776478>]

1. HPV type 16 E6 and NFX1-123 augment JNK signaling to mediate keratinocyte differentiation and L1 expression.Here, we identify a role for the protein NFX1-123 in regulating keratinocyte differentiation and events of the late HPV life cycle. Altogether, these studies define a role for NFX1-123 in mediating epithelial differentiation through the JNK signaling pathway, potentially linking expression of cellular genes and HPV genes during differentiation.

[<https://www.ncbi.nlm.nih.gov/pubmed/30903928>]

Day 5 - EGFP Gene Editing

Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within.

The purpose of the experiment is to understand growing liquid culture of E. coli. A well-grown colony from the LB-agar plate can directly be utilized for plasmid isolation. This is more convenient when you need to screen a large number of colonies. So if we have enough well-grown colonies, we can single colon a good one from the LB flat colonies.

Purpose of small shake culture - Bacterial liquid cultures should be shaken always (but not too vigorously), in order to ensure aeration and oxygen and nutrient availability as well as to avoid bacterial settlement on the flask bottom which would result in cell death from the lack of nutrient availability. Also, shaking prevents bacterial clumps or biofilm formation, ensuring prolific bacterial reproduction. Depending on the type of bacteria and the size of the flask, adjust rpm so that it's not too vigorous that it will damage the cells and prevent proper replication.

Procedure

1. Single colon the LB flat colonies that grown into the LB medium, make small shake culture.

Day 5 -NFX1-123 Gene Editing

Procedures:

1. Inculcate the linked output of previous day into Escherichia coli DH5a
2. Recovery Escherichia coli DH5a, spread evenly after centrifugal onto Solid medium (Ampicillin resistance)

Day 6 - EGFP Gene Editing

Procedures:

1. Extract the plasmatic from tremendously populated Escherichia coli from small shack, and do examine operation.

Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing.
Escherichia coli-based cell extract is a vital component of inexpensive and high-yielding cell-free protein synthesis reactions. However, effective preparation of E. coli cell extract is limited to high-pressure (French press-style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. Here we report the viability of E. coli cell extract prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples.
[<https://www.future-science.com/doi/full/10.2144/0000113924>]

Day 6 –NFX1-123 Gene Editing
Procedure

1. Single colon the LB flat colonies that grown into the LB medium, make small shake culture.

Day 6 – Cell experiment
Transient transfection exerts a temporary influence on your cells, whereas stable transfection leads to permanent genetic changes that are usually passed on to future cell progeny. So in our experiment we use transient transfection. The purpose of the experiment is to prepare for the later on WB, IP and MS experiments.

Procedure

1. Transient NFX1 protein into Hela cell to do Protein overexpression

Transient transfection technics researches are blow.
Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature. Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) .
[<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404257/>]

Transfection of Mammalian Cells with Fluorescent Protein Fusions.
Fluorescent protein fusions (FPFs) have been used to address a wide range of questions in individual cells as well as in specific tissues of particular organisms. However, investigators must take extreme care when using FPFs to ensure that the resultant fusion protein is expressed
[<http://cshprotocols.cshlp.org/content/2010/11/pdb.prot5517.full>]

Choosing a Transfection Strategy
Deciding whether you need transient or stable transfection depends on the time frame and ultimate goal of the experiment you wish to conduct. A major advancement in transient expression technology for rapid and ultra-high-yield protein production in mammalian cells is the Expi293™ Expression System.
[<https://www.thermofisher.com/cn/zh/home/references/gibco-cell-culture-basics/transfection-basics/choosing-transfection-strategy.html>]

Day 8 – EGFP Gene Editing
The purpose of this experiment is verifying the Insert Sequence with Sanger Sequencing. After identifying a few positive clones, the last step is to mini-prep these clones and submit the plasmids for Sanger sequencing. Sequencing allows you to confirm the sequence of the insert, insert orientation and the sequences of the junctions between the plasmid and insert DNA. .

Procedure

1. Small extraction plasmid to verify striped plasmids, then send to do DNA sequencing and more measurement

DNA Sequencing is so important and popular for Gene Technology that there are many DNA Sequencing services companies. Some examples are blow.
QIAGEN offers a wide range of sequencing services. Our single-read services enable routine sequencing of plasmids, PCR products, and expressed sequence tags (ESTs).
[<https://www.qiagen.com/us/products/discovery-and-translational-research/genomic-services/nextgenerationsequencing/dnasequencing/qiagen-sequencing-services/#orderinginformation>]

DNA Sequencing Sample Submission Guidelines
We accept PCR products (purified or unpurified), and plasmid DNA samples, and bacterial colonies or glycerol stocks.
[<https://www.genscript.com/sequencing_guidelines.html>]

Day 8 – NFX1-123(Mlu1) Gene Editing
MCB is Mlu1 Cell Cycle Box (anti-cancer genetic entity), Mlu1 is restriction enzymes. So this experiment is to use NFX1-123 restriction enzymes Mlu1.

Some commercial application usage of Mlu1 is below:
Thermo Scientific FastDigest MluI is one of an advanced line of fast restriction enzymes that are all 100% active in the universal FastDigest and FastDigest Green reaction buffers. The universal buffer allows rapid single-, double-, or multiple DNA digestion within 5–15 minutes eliminating any need for buffer change or subsequent DNA clean-up steps.
[<https://www.thermofisher.com/order/catalog/product/FD0564>]

Procedure

1. Extract the plasmids for verification out from the massive grown Escherichia coli colonies from small shake culture.

Day 8 – NFX1-123(Spe1) Gene Editing

A restriction enzyme (or restriction endonuclease) recognizes a specific nucleotide-pair sequence in DNA called a restriction site and cleaves the DNA (hydrolyzes the phosphodiester backbones) within or near that sequence.

The experiment use NFX1-123 SpeI,  a restriction endonuclease used for molecular biology applications to cut DNA at the recognition site 5′-A/CTAGT-3′, generating DNA fragments with 5′-cohesive ends, as shown in below picture. Then freeze the gel for later experiment.

![Picture 1](https://igem.siqint.com/hpv/images/index_clip_image002_0000.jpg)

Procedure

1. Pull out of a template the target Gene NFX1 using PCR method
2. Assess for the PCR results with agar sugar gel electrophoresis
3. Cut glue frozen in -20 refrigerator for later to use

Day 8 –WB Collecting Cells

A restriction enzyme (or restriction endonuclease) recognizes a specific nucleotide-pair sequence in DNA called a restriction site and cleaves the DNA (hydrolyzes the phosphodiester backbones) within or near that sequence.

Western Blot (WB) is a common method to detect and analyze proteins. It is built on a technique that involves transferring, also known as blotting, proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically.

WB is frequently performed in conjunction with other test methodologies and is very useful in providing additional test specificity, for example in the case of testing for HIV or Lyme disease.
[<http://www.imugen.com/test-methods/western-blot/>]

For our HPV cervical cancer research, it is a perfect tool to use and get very familiar with. This experiment goal is to learn to use WB method to collect cell and analyze proteins.

Procedure

1. 1X loading collecting cells, cook edgy, run glue after the turn film on NFX1 a resistance

Day 8 –IP Collecting Cells

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min.

EBC may be applied to the detection of lung cancer where it could be a tool in early diagnosis.

The experiment purpose is to learn to use EBC way to collect cells for later on cancer DNA research.

A The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu +2 to Cu +1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid.

Total protein quantitation methods comprise traditional methods such as the measurement of UV absorbance at 280 nm, Bicinchoninic acid (BCA) and Bradford assays, as well as alternative methods like Lowry or novel assays developed by commercial suppliers, which often provide a well-designed, convenient kit for each type of the assay.

One of the commercial application if from Quchem. The BCA Protein Assay is a detergent compatible formulation based on Bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein.
[<https://bioquochem.com/product/bca-protein-quantification-assay-kit-kb-03-005/>]

This experiment will learn the BCA protein assay as quantitation method.

Procedure

1. EBC collecting cells, after BCA quantification, wait for the quality protein to be placed in the new EP tube, co-incubate after joining Beads.

After cooking samples run glue for IP test dyeing, Input proceed to WB assess for expression effect.

Day 8 –MS-Total Collecting Cells

Filter-aided sample preparation (FASP) method is used for the on-filter digestion of proteins prior to mass-spectrometry-based analyses. FASP was designed for the removal of detergents, and chaotropes that were used for sample preparation. In addition, FASP removes components such as salts, nucleic acids and lipids.

This experiment will use FASP method to speed up the workflow, even though with increased costs .

Procedure

1. Ultrasound-cooked centrifugal extraction protein after SDT cell division
2. Tryptophan quantify, then remove same quality protein, FASP protease solution, add 1:50 ration, keep at temperature of 37 overnight for reaction.

Day 9 – EGFP Gene Editing
Day 8 experiment failed, redo the experiment.

Procedure

1. Small extraction plasmid to verify striped plasmids, then send to do DNA sequencing and more measurement

Day 9 – NFX1-123(Mlu1) Gene Editing
Day 8 experiment failed, redo the experiment.

Procedure

1. Extract the plasmids for verification out from the massive grown Escherichia coli colonies from small shake culture.

Day 9 – NFX1-123(Spe1) Gene Editing
Day 8 experiment failed, redo the experiment.

Procedure

1. Recovery of GFP target gene in the DNA gel
2. Double enzyme cut GFP target gene, nurture sticky end
3. Electrophoresis verification on enzyme extraction
4. Recovery of enzyme extraction to DNA gel
5. Determine the concentration of gel extraction
6. Link the carrier and target gene GFP base on Mole ratio 1:10.

Day 9 –WB Collecting Cells
Day 8 experiment failed, redo the experiment.

Procedure

1. Wash film, incubate two resistance, color exposure

Day 9 –IP Collecting Cells
Day 8 experiment failed, redo the experiment.

Procedure

1. Wash film, incubate two resistance, color exposure
2. Gel electrophoresis IP and Input Blotting
3. Remove the color of the IP blotting gel
4. Cut gel and Intra-gel enzyme solution

Day 9 –MS-Total Collecting Cells

Day 8 experiment failed, redo the experiment.

Procedure

1. Peptide segment
2. Freeze to dry and save at temperature -80 for later to use

Day 10 –MS-Total Collecting Cells

Day 9 experiment failed, redo the experiment.

Procedure

1. Extract the plasmids for verification out from the massive grown Escherichia coli colonies from small shake culture.

Day 10 –NFX1-123(Spe1) Gene Editing

Day 9 experiment failed, redo the experiment.

Procedure

1. Extract the plasmids for verification out from the massive grown Escherichia coli colonies from small shake culture.

Day 10 –WB Collecting Cells
Day 9 experiment failed, redo the experiment.

Procedure

1. Wash film, incubate two resistance, color exposure

Day 10 –IP Collecting Cells
Day 9 experiment failed at step #5, redo the experiment last step.

Procedure

1. Cut gel and Intra-gel enzyme solution

Day 11 –Test mass spectrometry on the peptide segment
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses.

This experiment goal is to digest the protein to peptides in DNA gel. Mass spectrometry gets limited sequence data from whole proteins, but can easily analyze peptides.

Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein.

Proteomics and Protein Mass Spectrometry Workflows
[<https://www.thermofisher.com/cn/zh/home/industrial/mass-spectrometry/proteomics-protein-mass-spectrometry/proteomics-protein-mass-spectrometry-workflows.html>]

Calibration Solutions, Standards, and Solvents for Mass Spectrometry
[https://www.thermofisher.com/cn/zh/home/life-science/protein-biology/protein-mass-spectrometry-analysis/calibration-solutions-standards-solvents-mass-spectrometry.html]

Day 11 – NFX1-123 (Spe1) Gene Editing
The experiment goal is to perform DNA sequencing on the verified striped plasmids.

DNA sequencing provides the most complete characterization of recombinant plasmid DNAs. Using primers targeting the plasmid backbone and/or the insert sequence, the identity and order of nucleotide bases for any given DNA can be determined.

Day 11 – Desalination of the enzymatic peptide segment
The experiment goal is to perform DNA sequencing on the verified striped plasmids.

Summary for NFX1-123 Gene Editing Experiment

The high-risk human papillomavirus (HR HPV) E6 interacts with the cellular protein NFX1-123, and together they post-transcriptionally increase hTERT expression, the catalytic subunit of telomerase.

Since HeLa cell line is a type of HPV cervical cancer cells, this implies that HeLa cells can express HPV L1 proteins. Background Human papillomavirus major capsid protein (HPV L1) is the main protein constituent of the HPV particle coat.

As a result, these Hela cells continuously overexpress target NFX-123 proteins (including monoclonal antibodies) and do not go into senescence. These are crucial in the production of antibodies, recombinant proteins, viral-subunit proteins, and vectors for gene therapy,

Particularly the NFX1 differential protein expression at the early stage of diagnostic for HPV cervical cancer, as well as therapeutic antibodies research is very crucial. That is the purpose for our NFX1-123 experiments.

The procedure of NFX1 experiments have 3 parts: molecule clone; cell comparison; FASP total protein MS.

Molecule Clone:

1. NFX1-123 PCR target gene in the DNA gel
2. Double enzyme cut target gene, nurture sticky end
3. Verify the PCR gel extraction through Agar sugar gel electrophoresis.
4. Single colon the LB flat colonies that grown into the LB medium, make small shake culture.
5. Test DNA sequence, compare results

Western Blot (WB) is a common method to detect and analyze proteins. It is built on a technique that involves transferring, also known as blotting, proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically. WB is frequently performed in conjunction with other test methodologies and is very useful in providing additional test specificity, for example in the case of testing for HIV or Lyme disease.

Cell Comparison:

1. Western Blot (WB) collecting cells, assess the cell cloned NFX1 protein through 1X loading collecting cells, cook edgy, run glue after the turn film on NFX1 a resistance
2. IP：EBC collecting cells, after BCA quantification, wait for the quality protein to be placed in the new EP tube, co-incubate after joining Beads. After cooking samples run glue for IP test dyeing, Input proceed to WB assess for expression effect.

Filter-aided sample preparation (FASP) method is used for the on-filter digestion of proteins prior to mass-spectrometry-based analyses. FASP was designed for the removal of detergents, and chaotropes that were used for sample preparation. In addition, FASP removes components such as salts, nucleic acids and lipids. This experiment will use FASP method to speed up the workflow.

FASP Total Protein MS

1. Ultrasound-cooked centrifugal extraction protein after SDT cell division
2. Tryptophan quantify, then remove same quality protein, FASP protease solution, add 1:50 ration, keep at temperature of 37 overnight for reaction.
3. Do the data analyzing for the Hela protein quality of Plex-EV verses Plex\_NFX1 to see the differential protein expression.

Based on the final data analyzing we can decide the possibility of the proposed HPV cervical cancer antibody effectiveness or the patient's early diagnosing of HPV cervical cancer.

Gel Electrophoresis of EGFP PCR
![](https://igem.siqint.com/hpv/images/5.1.png)
Figure-1: Gel Electrophoresis of EGFP PCR. All correct bands of EGFP are shown clearly. Gel extraction is applied.
![](https://igem.siqint.com/hpv/images/5.2.png)
Figure-2: Gel Electrophoresis of restriction fragment length polymorphism product of the EGFP gene. All bands of EGFP and NFX1 plasmid vectors are shown clearly. Gel extraction is applied.
![](https://igem.siqint.com/hpv/images/5.3.png)
Figure-3: Gel Electrophoresis of EGFP plasmid mini-prep. All correct bands of EGFP are shown clearly. Gel extraction is applied.
![](https://igem.siqint.com/hpv/images/5.4.png)

Figure-4:Gel Electrophoresis of NFX1-123 PCR. All correct bands of EGFP are shown clearly. Gel extraction is applied.

![](https://igem.siqint.com/hpv/images/5.5.png)
Figure-5:Gel Electrophoresis of restriction fragment length polymorphism product of the NFX1-123 gene. ⅘ bands of NFX1-123 are shown clearly in the correct size. Gel extraction is applied.

![](https://igem.siqint.com/hpv/images/5.6.png)
Figure-6:SDS-Pages of Hela cells transfected with NFX1-123 plasmid. The flow-through and elution fraction Empty Vectors and NFX1-1-3 are shown.![](https://igem.siqint.com/hpv/images/5.7.png)
Figure-7:
![](https://igem.siqint.com/hpv/images/5.8.png)

Figure 8:SDS-Pages of Hela cells transfected with NFX1-123 plasmid. The flow-through and elution fraction Empty Vectors and NFX1-1-3 are shown. The arrow marks the NFX1-123 band with a molecular weight of 123 KDA

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# Team:Shanghai-United/Experiments

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  + [Description](https://2019.igem.org/Team%3AShanghai-United/Description)
  + [Design](https://2019.igem.org/Team%3AShanghai-United/Design)
  + [Experiments](https://2019.igem.org/Team%3AShanghai-United/Experiments)
  + [Model](https://2019.igem.org/Team%3AShanghai-United/Model)
  + [Notebook](https://2019.igem.org/Team%3AShanghai-United/Notebook)
  + [Results](https://2019.igem.org/Team%3AShanghai-United/Results)
  + [Improve](https://2019.igem.org/Team%3AShanghai-United/Improve)
  + [Contribution](https://2019.igem.org/Team%3AShanghai-United/Contribution)
* [Parts](https://2019.igem.org/Team%3AShanghai-United/Parts)
* [Safety](https://2019.igem.org/Team%3AShanghai-United/Safety)
* [Demonstrate](https://2019.igem.org/Team%3AShanghai-United/Demonstrate)
* Team
  + [Attributions](https://2019.igem.org/Team%3AShanghai-United/Attributions)
  + [Team Member](https://2019.igem.org/Team%3AShanghai-United/Team)
* Human Practice
  + [Social Context](https://2019.igem.org/Team%3AShanghai-United/Social)
  + [Integrated HP](https://2019.igem.org/Team%3AShanghai-United/Human_Practices)
  + [Fundraising](https://2019.igem.org/Team%3AShanghai-United/Fundraising)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AShanghai-United/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3AShanghai-United/Collaborations)

The characterization protein and early diagnosis of cervical cancer

Here we demonstrate specific protein expression variations related to NFX1 over-expression, a gene found to be closely associated with cervical cancer.

### Experiment

**[1、 molecular cloning](https://igem.siqint.com/pdf/molecular%20cloning.pdf)**

**[2、 Cell experiments+Western Blot](https://igem.siqint.com/pdf/molecular%20cloning.pdf)**

**[3、 IP](https://igem.siqint.com/pdf/IP.pdf)**

**[4、 fasp](https://igem.siqint.com/pdf/fasp.pdf)**

©2019 wiki-hpv Corporation. All rights reserved.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Shanghai-United/Results

Home

![](https://igem.siqint.com/hpv/images/logo.png)

* [Home](https://2019.igem.org/Team%3AShanghai-United/)
* Project
  + [Description](https://2019.igem.org/Team%3AShanghai-United/Description)
  + [Design](https://2019.igem.org/Team%3AShanghai-United/Design)
  + [Experiments](https://2019.igem.org/Team%3AShanghai-United/Experiments)
  + [Model](https://2019.igem.org/Team%3AShanghai-United/Model)
  + [Notebook](https://2019.igem.org/Team%3AShanghai-United/Notebook)
  + [Results](https://2019.igem.org/Team%3AShanghai-United/Results)
  + [Improve](https://2019.igem.org/Team%3AShanghai-United/Improve)
  + [Contribution](https://2019.igem.org/Team%3AShanghai-United/Contribution)
* [Parts](https://2019.igem.org/Team%3AShanghai-United/Parts)
* [Safety](https://2019.igem.org/Team%3AShanghai-United/Safety)
* [Demonstrate](https://2019.igem.org/Team%3AShanghai-United/Demonstrate)
* Team
  + [Attributions](https://2019.igem.org/Team%3AShanghai-United/Attributions)
  + [Team Member](https://2019.igem.org/Team%3AShanghai-United/Team)
* Human Practice
  + [Social Context](https://2019.igem.org/Team%3AShanghai-United/Social)
  + [Integrated HP](https://2019.igem.org/Team%3AShanghai-United/Human_Practices)
  + [Fundraising](https://2019.igem.org/Team%3AShanghai-United/Fundraising)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AShanghai-United/Public_Engagement)
  + [Collaborations](https://2019.igem.org/Team%3AShanghai-United/Collaborations)

The characterization protein and early diagnosis of cervical cancer

Here we demonstrate specific protein expression variations related to NFX1 over-expression, a gene found to be closely associated with cervical cancer.

### Results

**[1、 MS\_Report](https://igem.siqint.com/pdf/MS_Report.pdf)**

**[2、 Important Proteins Page](https://igem.siqint.com/pdf/Important%20Proteins%20Page.pdf)**

**[3、 Differential protein Database](https://igem.siqint.com/pdf/Differential%20protein%20Database.pdf)**

©2019 wiki-hpv Corporation. All rights reserved.