iGEM 2024 — Team liuan-nanjing

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

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{% block title %}Engineering Success{% endblock %}
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# 1. Overview

Throughout the engineering design cycles of our project, team LIUAN-Nanjing designed and characterized 9 new basic parts and 8 new composite parts. We also provided a reversible 3’-O-(2-nitrobenzyl)-modified nucleotides to enable the ordered and controlled enzymatic synthesis of DNA.

Table 1: Parts Overview

| Part Number | Type | Description | Length |
| --- | --- | --- | --- |
| BBa\_K5392000 | Basic | ZaTdT-wild type | 1548bp |
| BBa\_K5392001 | Basic | ZaTdT-mutant type (ZaTdT-R335P) | 1548bp |
| BBa\_K5392002 | Basic | ZaTdT-mutant type (ZaTdT-R335W) | 1548bp |
| BBa\_K5392003 | Basic | ZaTdT-mutant type (ZaTdT-R335I) | 1548bp |
| BBa\_K5392004 | Basic | ZaTdT-mutant type (ZaTdT-R335F) | 1548bp |
| BBa\_K5392005 | Basic | ZaTdT-mutant type (ZaTdT-R335M) | 1548bp |
| BBa\_K5392006 | Basic | ZaTdT-mutant type (ZaTdT-K337A) | 1548bp |
| BBa\_K5392007 | Basic | ZaTdT-mutant type (ZaTdT-K337L) | 1548bp |
| BBa\_K5392015 | Composite | Vector-ZaTdT-KanR | 6832bp |
| BBa\_K5392016 | Composite | Vector-ZaTdT-R335P-KanR | 6832bp |
| BBa\_K5392017 | Composite | Vector-ZaTdT-R335W-KanR | 6832bp |
| BBa\_K5392018 | Composite | Vector-ZaTdT-R335I-KanR | 6832bp |
| BBa\_K5392019 | Composite | Vector-ZaTdT-R335F-KanR | 6832bp |
| BBa\_K5392020 | Composite | Vector-ZaTdT-R335M-KanR | 6832bp |
| BBa\_K5392021 | Composite | Vector-ZaTdT-K337A-KanR | 6832bp |
| BBa\_K5392022 | Composite | Vector-ZaTdT-K337L-KanR | 6832bp |

# 2. Engineering Cycle 1

## 2.1. Stage 1: Design—Selection Of TdT Orthologs And Nucleotides Modification

As a promising technology, enzymatic DNA synthesis has been gaining interest since the 1950s. Terminal deoxynucleotidyl transferase (TdT), one of the most promising DNA polymerases for de novo DNA
synthesis, has been known to be able to incorporate random nucleotides into initiator strands in the absence of a template since the 1960s[1-2]. However, the interest in developing enzymatic DNA
synthesis declined after the successful establishment of the phosphoramidite method during the 1980s. In
recent years, TdT has again attracted considerable attention due to the need for faster synthesis of longer DNA strands for data storage and synthetic biology[3-5].

The major challenge in the application of TdT for the programmed synthesis of artificial DNA lies in controlling the polymerization of user-defined nucleotides. Currently, reversible nucleotide
terminators have been modified to control DNA synthesis[6]. Among the functional modifications, reversible chemical moieties capping the 3’-OH group of the sugar unit have garnered great interest
due to their better termination effect[7]. However, TdT has a relatively small active cavity, making it
more difficult to incorporate sugar modified nucleotides than natural nucleotides[8-9]. The successful incorporation of sugar-modified nucleotides requires extensive engineering of TdT[10].
Therefore, the implementation of a 3’-O-blocked reversible terminator and TdT for de novo DNA synthesis relies on the selection of a suitable blocking group, engineering of TdT, and optimization
of cycling conditions.

The strategy of employing TdT for DNA synthesis relies on the stepwise addition of desired nucleotides. This can be accomplished by adopting a reversible terminator, which stop the synthesis
following the addition of each new nucleotide analogue. A wide range of sugar-modified nucleotide terminators have been successfully applied to pause nucleotide incorporation in DNA
sequencing[11]. Here, we designed a 3’-O-(2-nitrobenzyl)-dNTP that is a 3’-O-blocked reversible terminator.

![](https://static.igem.wiki/teams/5392/engineering/1.webp)

Figure 1: 3’-O-(2-nitrobenzyl)-dNTP

The current knowledge regarding the structural features and physiologicalfunctions of template-independent DNA polymerases is primarily derived nucleotide addition by TdTs derived from different
species has remained elusive[12]. A recent study based on the phylogenetic tree of 137 TdT genes across the vertebrate taxa, selected 14 TdTs from major clades of vertebrates including fish,
amphibians, mammals, reptiles, and birds to test their catalytic activities in the incorporation of nucleotides
into single-stranded initiators.They found that ZaTdT from *Zonotrichia albicollisnhad* the highest polymerization activity for natural nucleotide among the tested TdTs. Therefore, we chose
ZaTdT for the subsequent experiments.

![](https://static.igem.wiki/teams/5392/engineering/2.webp)

Figure 2: Phylogenetic relationship of TdTs from representative vertebrates and characterization of the products of TdT-mediated primer extension reactions

## 2.2. Stage 2: Build—Plasmids Construction

We incorporated the sequences of the target gene into the pET28a vector. Then the vector plasmid was transfected into *E.coli* DH5-alpha competent cells for purification and amplification.
In this way, plasmids with the ZaTdT gene were obtained.

![](https://static.igem.wiki/teams/5392/engineering/3.webp)

Figure 3: Schematic of Recombinant Plasmid

## 2.3. Stage 3: Test—catalytic activity assay of ZaTdT.

We transfected the ZaTdT gene-bonded pET28a plasmid into *E.coli* BL21(DE3) competent cell. After overnight, Colonies were then picked and performs protein expression. We identified the ZaTdT expression by SDS-PAGE and tested the incorporation of modified nucleotides by purified ZaTdT with polyacrylamide gel electrophoresis.

![](https://static.igem.wiki/teams/5392/engineering/4.webp)

Figure 4: SDS-PAGE of ZaTdT. M:marker; 1-2: Total Protein; 3-6: ZaTdT

![](https://static.igem.wiki/teams/5392/engineering/5.webp)

Figure 5: Denaturing PAGE analysis of the incorporation of four 3’-O-(2-nitrobenzyl)-blocked nucleotides by ZaTdT

## 2.4. Stage 4: Learn

Through this cycle, we learned how to use SnapGene to construct plasmid and the entire process of protein expression and purification as well as catalytic activity testing. Polyacrylamide gel electrophoresis (PAGE) can be used to observe initiator strand extension. Our experiments showed that ZaTdT displayed the highest incorporation efficiency to 3’-O-(2-nitrobenzyl)-dTTP and Slightly activity to 3’-O-(2-nitrobenzyl)-dCTP. Furthermore, other 3’-O-blocked reversible terminator showed no
observable extension when used wild type ZaTdT.

# 3. Engineering Cycle 2

## 3.1. Stage 1: Design—Protein Engineering of ZaTdT

To make the active center more compatible with 3’-O-(2-nitrobenzyl)-modified nucleotides, we attempted to reshape the catalytic cavity using mutagenesis on the residues within 6 Å around
3’-O-(2-nitrobenzyl) and the base moiety. We predicted the 3D structure of ZaTdT by homology modelling and molecular docking it with 3’-O-(2-nitrobenzyl)-dATP. The results of molecular docking
indicate that residues Arg335 and Lys337 are closely related to the catalytic activity of ZaTdT.

![](https://static.igem.wiki/teams/5392/engineering/6.webp)

Figure 6: Structure of ZaTdT and molecular docking it with 3’-O-(2-nitrobenzyl)-dATP

Analysis of the molecular dynamics (MD) trajectories showed that R335 and its spatially neighboring residue K337 in ZaTdT contacted the triphosphate group through hydrogen bonds. We anticipate that
eliminating the hydrogen bonds binding force between the above residues and the triphosphate group will release the greater freedom of the modified nucleoside within the active pocket, thereby
increasing catalytic activity. Consequently, We performed molecular dynamics simulations of the
3’-O-(2-nitrobenzyl)-dATP docked into ZaTdT, ZaTdT-R335P, ZaTdT-R335W, ZaTdT-R335I, ZaTdT-R335F, ZaTdT-R335M, ZaTdT-K337A and K337L. The predicted results show that above substitution can abolish
the hydrogen-bond connection at position 335 or reduce the frequency of hydrogen-bond formation between the triphosphate group and Lys337.

![](https://static.igem.wiki/teams/5392/engineering/7.webp)

Figure 7: Analysis of the molecular dynamics

## 3.2. Stage 2: Build—Plasmids Construction

To obtain the desired saturation mutagenesis, oligonucleotide primers were designed with degenerate codons. In addition, each single-site saturation mutant was generated according to the PCR-based
QuickChange method. The PCR was performed according to the operation manual. The PCR product was digested with DpnI restriction enzyme and transformed into *E.coli* DH5-alpha competent
cells.

## 3.3. Stage 3: Test—catalytic activity assay of ZaTdT Mutant.

We transfected the Sequencing is correct ZaTdT mutant plasmid into *E.coli* BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify
its activity.

![](https://static.igem.wiki/teams/5392/engineering/8.webp)

Figure 8: SDS-PAGE of ZaTdT mutants. M:marker; 1: ZaTdT-R335P; 2: ZaTdT-R335W; 3: ZaTdT-R335I; 4: ZaTdT-R335F; 5: ZaTdT-R335M; 6: ZaTdT-K337A; 7: ZaTdT-K337L.

![](https://static.igem.wiki/teams/5392/engineering/9-1.webp)
![](https://static.igem.wiki/teams/5392/engineering/9-2.webp)

Figure 9: Denaturing PAGE analysis of the incorporation of four 3’-O-(2-nitrobenzyl)-blocked nucleotides by ZaTdT mutants

## 3.4. Stage 4: Learn

Through this cycle, we learned how to use homology modelling to predict the 3D structure of protein and use molecular docking to excavate closely related residues to the catalytic activity of
protein. Molecular dynamics (MD) simulations can provide guidance for protein engineering. Elimination of hydrogen-bonding forces releases a larger nucleotide of freedom, which improves the
catalytic activity of ZaTdT. In this cycle, we obtained two mutants, ZaTdT-K337A and ZaTdT-K337L, that could extend all four 3’-O-blocked reversible terminators, after performing the reduce the
frequency of hydrogen-bond formation between the triphosphate group and Lys337. Ultimately, we found that BBa\_K5392000 was new basic part because it had better catalytic activity for natural dNTP
than other sources of TdTs and BBa\_K5392021 was new composite part, because they obtained better catalytic activity compared to wild-type ZaTdT or other ZaTdT mutant for 3’-O-(2-nitrobenzyl)-dNTP
that is a 3’-O-blocked reversible terminator.

# 4. Reference

[1] BOLLUM FJ. Thermal conversion of nonpriming deoxyribonucleic acid to primer. *J Biol Chem*. 1959;234:2733-2734.

[2] Bollum FJ. Chemically Defined Templates and Initiators for Deoxypolynucleotide Synthesis. *Science*. 1964;144(3618):560.

[3] Anavy L, Vaknin I, Atar O, Amit R, Yakhini Z. Data storage in DNA with fewer synthesis cycles using composite DNA letters. *Nat Biotechnol*. 2019;37(10):1229-1236.

[4] Hutchison CA 3rd, Chuang RY, Noskov VN, et al. Design and synthesis of a minimal bacterial genome. *Science*. 2016;351(6280):aad6253.

[5] Mercy G, Mozziconacci J, Scolari VF, et al. 3D organization of synthetic and scrambled chromosomes. *Science*. 2017;355(6329):eaaf4597.

[6] Minhaz Ud-Dean SM. A theoretical model for template-free synthesis of long DNA sequence. *Syst Synth Biol*. 2008;2(3-4):67-73.

[7] Chen F, Dong M, Ge M, et al. The history and advances of reversible terminators used in new generations of sequencing technology. *Genomics Proteomics Bioinformatics*. 2013;11(1):34-40.

[8] Loc'h J, Rosario S, Delarue M. Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination. *Structure*. 2016;24(9):1452-1463.

[9] Delarue M, Boulé JB, Lescar J, et al. Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase. *EMBO J*. 2002;21(3):427-439.

[10] Sosunov VV, Santamaria F, Victorova LS, Gosselin G, Rayner B, Krayevsky AA. Stereochemical control of DNA biosynthesis. *Nucleic Acids Res*. 2000;28(5):1170-1175.

[11] Guo J, Yu L, Turro NJ, Ju J. An integrated system for DNA sequencing by synthesis using novel nucleotide analogues. *Acc Chem Res*. 2010;43(4):551-563.

[12] Gouge J, Rosario S, Romain F, Beguin P, Delarue M. Structures of intermediates along the catalytic cycle of terminal deoxynucleotidyltransferase: dynamical aspects of the two-metal ion mechanism. *J Mol Biol*. 2013;425(22):4334-4352.

[13] X Lu, J Li, C Li, et al. Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase. *ACS Catalysis*. 2022; 12(5):2988-2997.

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{% block title %}Experiments{% endblock %}
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# 1. Recovering Plasmid DNA

Materials:

1. Overnight culture of bacteria transformed with plasmid
2. Plasmid Mini Kit
3. Sterilized distilled water

Equipment:

1. Eppendorf tube
2. Desktop microcentrifuge
3. Desktop vortexer
4. thermostat water bath

Procedures:

1. Grow an overnight culture of bacteria.
2. Centrifuge the 1~5mL culture to pellet the bacteria before proceeding with DNA preparation.
3. Remove the supernatant and resuspend the bacteria in buffer.
4. Add a denaturing solution from the Plasmid Mini Kit to the resuspended bacteria.
5. Add a renaturing solution from the Plasmid Mini Kit to the denatured bacteria.
6. Pellet the proteins and genomic DNA by centrifugation and remove the plasmid-containing supernatant.
7. Add ethanol to precipitate the plasmid DNA.
8. Centrifuge at 12000rpm for 10 min to pellet the DNA.
9. Wash the pellet with 70% ethanol to remove excess salt.
10. Resuspend the DNA pellet Sterilized distilled water.

# 2. Diagnostic Endonuclease Digest

Materials:

1. Plasmid DNA
2. Restriction enzyme
3. Restriction digest buffer
4. Forward Primer
5. Reverse Primer
6. 10X PrimeSTAR GXL Buffer with MgCl2
7. dNTP mix
8. PrimeSTAR GXL DNA Polymerase

Equipment:

1. Thin-walled PCR tube
2. Ice box
3. PCR Machine

Procedures:

1. In a 1.5mL tube combine the following:
   1. 1μg of plasmid DNA sample
   2. 1μL of each restriction enzyme
   3. 3μL restriction digest buffer
   4. H2O up to total volume of 30μL
2. Mix gently by pipetting.
3. Incubate tube at 37°C for 1hour.
4. Place thin-walled PCR tubes on ice. Set up a 50μL reaction:
   1. 1μL template DNA
   2. 5μL 10X PrimeSTAR GXL Buffer with MgCl2
   3. 4μL dNTP mix
   4. 2μL of 10μM Forward Primer
   5. 2μL of 10μM Reverse Primer
   6. 1μL PrimeSTAR GXL DNA Polymerase
   7. H2O up to total volume of 50μL
5. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:
   1. Initial Denaturation for 2min at 98°C.
   2. Denature for 10s at 98°C.
   3. Anneal primers for 30s at 55°C.
   4. Extend DNA for 1min at 72°C.
   5. Repeat b-d steps for 20-30 cycles.
   6. Extension for 5 min at 72°C.
6. The products were stored at 4 °C.

# 3. Acquisition of Target Gene Fragment

Materials:

1. 10X PrimeSTAR GXL Buffer with MgCl2
2. template DNA
3. Forward Primer
4. Reverse Primer
5. dNTP Mixture
6. PrimeSTAR GXL DNA Polymerase
7. Sterilized distilled water

Equipment:

1. Eppendorf tube
2. Desktop microcentrifuge
3. Desktop vortexer
4. Ice box
5. PCR instrument

Procedures:

1. Place thin-wall PCR tubes on ice. Set up a 50μL reaction:
   1. 1μL template DNA
   2. 5μL 10X PrimeSTAR GXL Buffer
   3. 4μL dNTP mix
   4. 2μL of 10μM Forward Primer
   5. 2μL of 10μM Reverse Primer
   6. 1μL PrimeSTAR GXL DNA Polymerase
   7. H2O to a total of 50μL
2. Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:
   1. Initial Denaturation for 2min at 98°C.
   2. Denature for 10 seconds at 98°C.
   3. Anneal primers for 15s at 55°C below.
   4. Extend DNA for 1min at 72°C.
   5. Repeat b-d steps for 30 cycles
   6. Extension for 5 min at 72°C
   7. The products were stored at 4°C.

# 4. Construction of Recombinant Plasmids

Materials:

1. Linearized vector plasmid DNA
2. Target gene segment
3. DNA ligase and buffer
4. Competent state *Escherichia coli* DH5α
5. Luria-Bertani media

Equipment:

1. Eppendorf tube
2. Shaking incubator at 37°C
3. Stationary incubator at 37°C
4. Water bath at 42°C
5. Ice box
6. Microcentrifuge tubes
7. Sterile spreading device

Procedures:

1. Combine the following in an Eppendorf tube:
   1. 2.5μL Plasmid Vector DNA
   2. 2μL Insert DNA (For a control group, add H2O instead of insert DNA)
   3. 2μL Ligase Buffer
   4. 1μL DNA Ligase
   5. H2O to a total of 20μL
2. Incubate at 37°C for 30min before cooling in an ice water bath for 5min.
3. Retrieve competent cells from -80°C refrigerator and defrost on ice for approximately 10minutes.
4. Retrieve the agar plates containing the appropriate antibiotic from storage at 4°C, and let them warm up to room temperature.
5. Mix 10μL of plasmid DNA into 100μL of competent cells in a microcentrifuge tube. Gently mix the components. For a control group, add 10μL of water to verify that the LB agar’s antibiotic functions properly.
6. Incubate the competent cell/DNA mixture on ice for approximately 30minutes.
7. Heat shocks each transformation tube by placing the bottom half of the tube into a 42°C water bath, leaving it for approximately 45s.
8. Put the tubes back on ice for 2min.
9. Add 300μL LB media (without antibiotic) to the bacteria, and grow in 37°C shaking incubator for 30 min.
10. Plate an appropriate amount of the transformation onto the LB agar plate containing the corresponding antibiotic. Incubate plates at 37°C overnight.

# 5. Agarose Gel Electrophoresis

Materials:

1. DNA samples
2. Gel loading dye
3. 10X TAE bufferl
4. Agarose
5. 4S green plus

Equipment:

1. Well combs
2. Gel box
3. Microwave
4. Electrophoresis chamber
5. Voltage source
6. UV light source
7. Casting tray

Procedures:

1. Mix 0.2g of agarose powder and 1μL of 4S green plus with 20mL 1XTAE in a microwavable flask.
2. Microwave for 1-3 min until the agarose is completely dissolved.
3. Let agarose solution cool down to about 50°C in about 5 min.
4. Pour the agarose into a gel tray with the well comb in place.
5. Place the gel at room temperature for 20-30 min, until it has completely solidified.
6. Place the gel into the gel box of electrophoresis unit. Fill gel box with 1xTAE until the gel is covered.
7. Carefully load a molecular weight ladder.
8. Add loading buffer to each DNA sample. Carefully load samples into the additional wells of the gel.
9. Run the gel at 120 V for approximately 15 min.
10. Turn off power, disconnect the electrodes, and remove the gel from the gel box.
11. Visualize DNA fragments using UV light source.

# 6. Recombinant Protein Expression

Materials:

1. Recombinant *Escherichia coli* BL21
2. Antibiotic
3. 1M Isopropyl-β-D-thiogalactopyranoside（IPTG）
4. Luria-Bertani media

Equipment:

1. 50mL shaking flask
2. Shaking incubator at 37°C
3. Shaking incubator at 18°C
4. Stationary incubator at 37°C

Procedures:

1. Pick a single colony from the plate and add it to 10mL of LB medium, incubate at 37°C overnight with shaking.
2. 5mL of the above bacteria solution was added to 1L1L of LB medium, incubated at 37°C with shaking until the OD600 was between 0.40~0.45, and left to stand at room temperature for 45min.
3. incubate at 18°C for 45min.
4. Add 200uL of 1M IPTG solution and incubate at 18°C for 16~18h.
5. Centrifuge at 4°C and discard the supernatant to collect the bacteria..

# 7. Extraction and Verification of Protein

## 7.1. Extraction of Protein

Materials:

1. Recombinant *Escherichia coli* BL21
2. Lysis buffer
3. Elution buffer
4. SDS buffer

Equipment:

1. Eppendorf tube
2. 50mL Centrifuge tube
3. HisCap IDA 6FF
4. Prepacked Desalting Column
5. Amicon® Ultra
6. Desktop centrifuge
7. Metal bath pot
8. Centrifugal machine
9. Ultrasonic cell breaker
10. ÄKTA pure

Procedures:

1. Add 40mL Lysis buffer to the cell, resuspend the cells, and ultrasonically break the cells on ice for 1h.
2. After centrifugation at 11000rpm for 40min at 4°C, the supernatant was collected and passed through a 0.22um filter membrane.
3. Separate the protein samples using ÄKTA pure and combine the target proteins according to the UV absorption peaks.
4. Imidazole was removed from the solution by Prepacked Desalting Column.
5. Concentrate the protein using Amicon® Ultra and store at -80°C in a refrigerator.

## 7.2. Verification of Protein

Materials:

1. SDS-PAGE gel
2. SDS-PAGE running buffer
3. Loading buffer
4. Protein Ladder
5. Coomassie brilliant blue G-250
6. Coomassie Blue Staining Destaining Solution

Equipment:

1. SDS-PAGE electrophoresis tank
2. SDS-PAGE transfer tank
3. Microwave
4. Imager System

Procedures:

1. The SDS-PAGE gel was placed in the electrophoresis tank. Connect electrode and load 20µL of each sample into the gel.
2. Run the gel at 100 V for approximately 20 minutes, until the dye has migrated into the running gel.
3. Increase the voltage to 150 V and continue for approximately 30 minutes, until the dye front has reached the bottom of the gel.
4. Place the gel into a small tray. Add Coomassie brilliant blue G-250 and heat for 1 min.
5. Pour off the Coomassie brilliant blue G-250 and add dehydration solution for rinsing.
6. Put the gel into the imager and select the appropriate exposure time, and save the image.

# 8. Functional Verification of protein

Materials:

1. Protein
2. modified nucleotides
3. Oligonucleotides
4. PBS
5. CoCl2 solution
6. TBE buffer
7. SYBR™ Gold
8. Sterilized distilled water

Equipment:

1. Thin-walled PCR tube
2. Ice box
3. PCR Machine

Procedures:

1. Catalytic activity of protein was determined in 30µL reaction mixtures.
2. The reaction system comprised 1uM Oligonucleotides, 0.1 mM dNTPs, 0.25mM CoCl2 and PBS.
3. The catalytic reaction was started by adding 0.5 mg/mL purified protein.
4. After incubation at 30 °C for 20 min, the reaction was stopped by heating at 95°C.
5. The products of the reaction were analyzed by gel electrophoresis on a 20% acrylamide gel and 8M urea.
6. Subsequently, the PAGE gel was stained with SYBR™ Gold nucleicacid gel dye for 20 minutes and scanned with a fully automatic fluorescence image analysis system.
7. The result was processed with Gel Image System analysis software.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project and what you learned from them. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}