iGEM 2023 — Team gec-china

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{% block title %}Notebook{% endblock %}
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File 1. Lab Notebook.

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## ![My Icon](https://static.igem.wiki/teams/5005/wiki/svg/engineering-design.png) Design

Directed evolution techniques not only facilitate the rapid optimization of proteins, but also enable the optimization of specific cell types or their genetic backgrounds, providing a platform for developing targeted drugs. In 2018, for her outstanding contributions in the field of "directed evolution of enzymes", Professor Frances H. Arnold was awarded the Nobel Prize in Chemistry.

![](https://static.igem.wiki/teams/5005/wiki/ens/ens-fig1.jpg)

Figure 1. Prof. Frances H. Arnold wins the 2018 Nobel Prize in Chemistry.

The
basic process of directed evolution can be divided into three main steps:
(1) Constructing a mutant library.
(2) Expressing the library in a suitable expression system.
(3) Performing fast, efficient, and high-throughput selection or screening.
In sum, directed evolution aims to simulate natural evolution under specific laboratory conditions.
By inducing mutations and recombination, a large library of mutants is created. After expressing the mutations in host cells, specific conditions are used for selection or screening to identify proteins with desired characteristics, achieving molecular-level evolution simulation.

![](https://static.igem.wiki/teams/5005/wiki/ens/ens-fig2.jpg)

Figure 2. The schematic illustration for Directed Evolution.

## ![My Icon](https://static.igem.wiki/teams/5005/wiki/svg/engineering-build.png) Build

To construct a mutation library, a recently developed system called TRACE (T7 polymerase-driven continuous editing) has been introduced in recent years. This system utilizes AID (activation-induced cytidine deaminase) fused with T7 RNA polymerase (T7 RNAP) as the editor, enabling continuous and targeted mutations in mammalian cells. When this system is activated, the editor recognizes the target gene sequence containing the T7 promoter and introduces random mutations on the target gene through cytidine deaminase during transcription. During the preparation of our iGEM competition, Professor Yang Yu from the Institute of Biophysics, Chinese Academy of Sciences introduced this powerful system to us, which has been extensively used in his lab, and told us we are welcome to harness this tool to achieve our goal to improve the activity of NAD+ production enzymes.

![](https://static.igem.wiki/teams/5005/wiki/ens/ens-fig3.png)

Figure 3. The schematic illustration for the TRACE System.

Therefore, we first obtained the cell line that was pre-built with the Editor system from Professor Yang Yu's laboratory. This cell line incorporates the AID-T7 RNAP-UGI Editor system inducibly expressed by doxycycline (Dox). After expression, this system can target and induce mutations in the target gene (Target) driven by the T7 promoter.

## ![My Icon](https://static.igem.wiki/teams/5005/wiki/svg/engineering-test.png) Test

Based on previous research, the coding sequence of the blue fluorescent protein (BFP) gene can be used to validate the feasibility of the TRACE mutation system visually. This BFP gene is derived from the EGFP gene, with a mutation of the 199th base from T-A to C-G, shifting the fluorescence color from green to blue.Therefore, if the TRACE system is feasible, the mutated BFP gene obtained from site-directed mutagenesis can be reversed back to the EGFP gene, resulting in a shift in cell fluorescence from blue to green.
The following experiments were co-performed under the guidance of our advisors. To obtain the pTarget-EBFP plasmid, we first cloned the EGFP gene and linearized the pTarget plasmid using restriction endonucleases. Subsequently, we performed homologous recombination to ligate the linearized pTarget vector with the EGFP gene, resulting in the successful generation of the pTarget-EGFP plasmid with correct sequencing. Next, using a pair of site-directed mutagenesis primers, we introduced a mutation at nucleotide position 199 in the EGFP gene, changing T to C. We ultimately succeeded in constructing the pTarget-EBFP plasmid with correct sequencing (sequencing results specifically showed the bases near the site of the site-directed mutation, with the remaining sequences confirmed to be accurate).

![](https://static.igem.wiki/teams/5005/wiki/ens/ens-fig5.png)

Figure 4. The sequence results for the construction of EGFP and EBFP plasmids.

Then, we introduced this BFP plasmid into the cell line that was pre-built with the Editor system as mentioned above. We proceeded to activate the TRACE system by adding doxycycline at a final concentration of 1μM to the culture medium. This induced the expression of AID-T7 RNAP-UGI, initiating continuous mutagenesis of the target gene. In the BFP validation experiment, we observed significant green fluorescence after 8 days of doxycycline induction, confirming the feasibility of the TRACE system.

![](https://static.igem.wiki/teams/5005/wiki/ens/ens-fig4.png)

Figure 5. The immunofluorescence results confirmed the successful directed evolution of GFP from BFP by the TRACE system.

## ![My Icon](https://static.igem.wiki/teams/5005/wiki/svg/engineering-learn.png) Conclusion

This exciting success was not achieved overnight, but rather through many failures and subsequent troubleshooting and retries. For example: (1) Initially, we hoped to directly transform the wild-type BFP protein into GFP protein using the directed evolution system. It became apparent that more mutations would be required, and these mutations needed to occur simultaneously on one mRNA in order for it to be successful. Due to very low probability, it did not succeed. (2) After dismissing the first approach, we started thinking about how to use a simpler system to validate the possibility of our directed evolution. This process was time-consuming and demanding, requiring full support and a literature search from our advisory team (3) During the process of finding and confirming the final feasible approach, we also encountered many problems, such as the expressed BFP protein not fluorescing, introducing new mutations through plasmid construction, and the directed evolution time being too short, resulting in no green fluorescence appearing.
 Fortunately, through the relentless efforts of team members and guidance from our advisors, we finally saw the exciting green fluorescence, which proved the feasibility of this system and the engineering success required for our next experimental goal.

# Experiment 1

# Experiment purpose: Insert the NAMPT gene into a mammalian expression vector (pCDH) to express the NAMPT protein

# Method: Molecular cloning

![](https://static.igem.wiki/teams/5005/wiki/protocol/protocol-fig1.png)

# STEP 1: Get the CDS sequence of the NAMPT gene from NCBI

# STEP 2: Use SnapGene to construct the new plasmid

# STEP 3: PCR reaction

|  |  |
| --- | --- |
| Reagent | Volume ( µL) |
| PCR grade water | 21 |
| KOD One™ PCR Master Mix | 25 |
| 10 µM Primers Forward/Reverse | 1.5 |
| Template | 1 |
| Total volume | 50 |

PCR cycle

1) Denaturation: Break the hydrogen bonds between DNA bases
to melt the DNA double strands to produce single-stranded DNA.

2) Annealing: Lower the temperature low enough to allow
the oligonucleotide primer to bind to the DNA template.

3) Extension: During extension, DNA polymerase will
synthesize new double-stranded DNA.

|  |  |  |  |
| --- | --- | --- | --- |
| Process | Temperature(℃) | Time(s) | Cycle times |
| Pre-denaturation | 98 | 180 | 1 |
| denaturation | 98 | 10 | 30 |
| Annealing | 65 | 5 |
| Elongation | 68 | 20 |
| Elongation 2 | 65 | 300 | 1 |
| preservation | 4 | - | 1 |

# STEP 4: Empty vector double digestion for linearization

Reaction mix: 20ul system

Ingredients:

|  |  |
| --- | --- |
| Reagent | Volume ( µL) |
| pCDH vector | 3 |
| BamHl | 0.5 |
| EcoRI | 0.5 |
| ddH2O | 16 |
| Total volume | 20 |

Condition: PCR tube, 37℃, overnight

# STEP 5: Carrier double digestion linearization

1. Weigh 0.3g agarose powder and add it to 30 mL TAE
solution. Microwave oven to melt the agarose powder until the solution is clear and transparent.

2. Pour the agarose gel solution into the gluing tank and
insert the comb.

3. After solidification for about 30 minutes, take out
the nucleic acid glue and use or store it in TAE solution immediately for later
use.

# STEP 6: Agarose Gel Electrophoresis

Observe nucleic acid gel electrophoresis.

# STEP 7: Gel extraction

1.
Cut gel to 1.5ml EP tube.

2.
Add NT1 buffer (200 µl/mg).

3.
Dissolve the gel in the buffer in a metal bath at 50℃ for 5-10min

4.
Load the sample into the column.

5.
Use a centrifuge (11000xg,30s) to centrifuge the DNA from the buffer.

6.
Change a tube for a column, add NT3 buffer (700 µl/mg).

7.
Use centrifugal machine (11000xg,30s) to centrifuge the DNA from the buffer.

8. Repeat steps 6 and 7.

9.
Dry silica membrane (11000xg, 1min).

10. Change tube and add 50 µl ddH₂O, then elute the
DNA into the ddH₂O by Centrifuge (11000xg,30s).

# STEP 8: Concentration determination

1.
Add 2 µl to the NanoDrop 2000.

2.
Determinate concentration.

# STEP 9: The carrier is connected to the fragment

1. Mix Enzyme mix (5 µl), Cut vector (1 µl), NAMPT fragment
(3 µl), ddH2O(1 µl)

2. Incubate at 50℃ for
15 min

(Immediate use it for transformation; If subsequent
operations cannot be carried out immediately, the reaction sample should be
stored at -20℃)

# STEP 10: Transformation

1.
Operation Method

1) Take 50 µL of competent cells
melted in an ice bath, add the target DNA, mix them gently, and place in an ice
bath for 30 minutes.

2) Heat in a 42℃ water bath for 45 seconds, then quickly
transfer the tube to an ice bath for 2 minutes without
shaking the centrifuge tube.

3) According to the experimental requirements
(transformation of plasmids and recombinant ligation products), pipette different
volumes of transformed competent cells and add them to LB agar medium
containing corresponding antibiotics, and spread the cells evenly. Place the
plate at 37℃ until the liquid is absorbed, invert
the plate, and culture overnight at 37℃.

2.
Precautions

1) Freshly thawed cells have the highest transformation
efficiency.

2)
Avoid repeated thawing.

3)
Avoid pipetting.

4) The whole operation should be
gentle.

# STEP 11: Colony PCR-monoclonal identification

1. Picked a single colony into the medium containing
100ul ampicillin resistance, cultured on a shaker at 37℃ for 1 hour.

2. Same as step 3, perform a PCR (10ul system).

3. Same as step 5+6, Agarose
Gel Electrophoresis.

4. Shake positive
colonies tube 7ml culture medium overnight culture.

5.
Plasmid extraction.

# STEP 12: Plasmid extraction

1. Column equilibration step: Add 500℃L of
equilibrium liquid BL to the adsorption column CP3 (the adsorption column is
placed in the collection tube), centrifuge at 12,000 pm (~13,400xg) for 1 min,
pour off the waste liquid in the collection tube, and remove the adsorption.
Return the column to the collection tube.

2. Take 1-5 mL of overnight cultured bacterial solution,
add it to a centrifuge tube, use a conventional desktop centrifuge, centrifuge
at 12,000 rpm (~13,400Xg) for 1 min, and suck up
the supernatant as much as possible (when there is a lot of bacterial
solution, you can centrifuge the bacteria several times).

3. Add 250µL of solution P1
(please check whether RNase A has been added) to the centrifuge tube with the
bacterial pellet left, and use a liquid dispenser or a vortex shaker to suspend
the bacterial pellet thoroughly. (If there are bacteria blocks that are not
thoroughly mixed, the lysis will be affected, resulting in low extraction
volume and purity).

4. Add 250µL solution P2 to the
centrifuge tube, and gently turn it up and down 6-8
times to fully lyse the bacteria (Mix gently, do not shake vigorously,
so as not to break the genomic DNA, resulting in genomic DNA fragments mixed in
the extracted plasmid. At this time, the bacterial solution should become clear
and viscous, and the time used should not exceed 5 minutes to avoid damage to
the plasmid. If it does not become clear, it may be due to too much bacteria
and incomplete lysis, and the amounts of bacteria should be reduced).

5. Add 350µL of solution P3 to
the centrifuge tube, turn it up and down gently 6-8 times immediately, and mix
well. At this time, a white flocculent precipitate
will appear. Centrifuge at 12,000 rpm (~13,400Xg) for 10 min. (P3 should be
mixed immediately after adding to avoid local precipitation. If there are tiny
white precipitates in the supernatant, centrifuge again and take the
supernatant).

6. Use a pipette to transfer the supernatant collected in
the previous step to the adsorption column CP3 (the adsorption column is placed
in the collection tube), and be careful not to suck
out the precipitate as much as possible. Centrifuge at 12,000 rpm
(~13,400Xg) for 30-60 sec, discard the waste liquid in the collection tube and
put the adsorption column CP3 into the collection tube.

7. Add 600µL of rinse solution
PW to the adsorption column CP3 (please check whether absolute ethanol has been
added first), centrifuge at 12,000 rpm (~13,400xg) for 30-60 sec, pour off the
waste liquid in the collection tube, and put the adsorption column CP3 into the
collection tube.

8.
Repeat step seven.

9. Put the adsorption column CP3 into the collection tube
and centrifuge at 12,000 rpm (13,00xg) for 2 minutes to remove the residual
washing liquid in the adsorption column. (The residual ethanol in the rinsing
solution will affect the subsequent enzyme reaction experiments. In order to
ensure that downstream experiments will not be affected by residual ethanol, it
is recommended to remove the cap of the
adsorption column CP3 and place it at room
temperature for several minutes to dry the residual rinse solution in
the adsorption material completely).

10. Put the adsorption column CP3 in a clean centrifuge
tube, add 50-100℃L elution buffer H2O dropwise to the middle part of the adsorption membrane, place℃
at room temperature for 2 minutes, and centrifuge at 12,000 rpm (-13,400Xg) for
2 minutes to remove the plasmid The solution was collected into a centrifuge
tube.

# Experiment 2

Experimental purpose:to construct the
mutant form of NAMPT selected by directed evolution.

Method:Site-directed mutagenesis

![ͼƬ1](https://static.igem.wiki/teams/5005/wiki/protocol/protocol-fig2.png)

# STEP 1 - Primer Design

Precautions

1. The primer length should be generally between 15-30
bases. Otherwise, it will not be conducive to the reaction of the polymerase.

1) Primers that are too short will reduce amplification
specificity.

2) On the other hand, if the primer is too long, the
annealing temperature will increase.

2. The GC content of the primer should be between 40% and
60%, and the Tm value is preferably close to 72℃.

1) Tm value: The Tm value (melting temperature) of the
primer = 4(G+C)+2(A+T), try to ensure that the Tm value of the upstream and
downstream primers is consistent, generally not exceeding 2℃.

3. The bases are supposed to be distributed randomly.

1) Avoid repeating nucleobases to cause false priming;
try to avoid complementarity between primers to prevent the formation of primer
dimers or hairpin structures.

4. Primer specificity: If the 3' end of the primer
contains a G or C residue, the specificity of the primer can be increased.

# STEP 2 - PCR Reaction

1.
Reaction mix

|  |  |
| --- | --- |
| Reagent | Volume ( µL) |
| PCR grade water | 7 |
| KOD One™ PCR Master Mix | 10 |
| 10 µM Primers F/R | 1 each |
| Template | 1 |
| Total volume | 20 |

2.
PCR cycle

|  |  |  |  |
| --- | --- | --- | --- |
| Process | Temperature (℃) | Time(s) | Cycle times |
| Pre-denaturation | 98 | 180 | 1 |
| denaturation | 98 | 10 | 22 |
| Annealing | 58 | 5 |
| Elongation | 68 | 90 |
| Elongation 2 | 65 | 300 | 1 |
| preservation | 4 | - | 1 |

# STEP 3 - Prepare Nucleic Acid Gel

1.       Weigh 0.3g of agarose powder, add it to 30ml TAE
solution, and heat it in a microwave oven to fully melt the agarose powder
until the solution is clear and transparent.

2.     Pour the agarose gel solution into the gel tank and
insert the comb.

3.      After about 30 min of solidification, take out the
nucleic acid gel and use it immediately or store it in TAE solution for later
use.

# STEP 4 - Nucleic acid gel electrophoresis

Take 10 µL of the PCR product
for gel identification, and check whether there is a single band at the
position corresponding to the size of the band, so as to infer whether the PCR
is successful.

# STEP 5 - Add the Dpnl enzyme digestion template

1.       Reaction mix

|  |  |
| --- | --- |
| Reagent | Volume ( µL) |
| water | 7 |
| 10 µL FlashOne™ buffer | 2 |
| DNA | 10 |
| FlashCut™ Dpnl | 1 |
| Total volume | 20 |

2.
Operating process

1) Preparation of reaction system.

2) Gently suck or flick the tube wall to mix well (do not
vortex), and then centrifuge instantaneously to collect the wall-hanging
droplets.

3) Incubate at 37℃ for 60min.

# STEP 6 - Transform

1. Take 50 µL of competent cells melted on an ice bath,
add the DNA of interest, mix gently, and leave in an ice bath for 30 min.

2. Heat in a water bath at 42 ℃ for 45 sec, then quickly
transfer the tubes to an ice bath for 2 min, the process without shaking the
centrifuge tubes.

3. Add 500 µL of sterile LB medium (without antibiotics)
to each centrifuge tube, mix well and place in 1 h aphine plates at 37 ℃, 200
rpm to revive the bacteria.

4. Pipette different volumes of transformed competent
cells onto LB agar medium containing the appropriate antibiotics, and spread
the cells evenly. Place the plate at 37 ℃ until the liquid is absorbed, invert
the plate and incubate at 37 ℃ overnight.

# STEP 7 - Pick multiple single clones for sequencing identification

1. Each
person picked 3 colonies, cultured them into a medium containing ampicillin
antibiotic, and incubated them at 37℃ over night.

# STEP 8 - Plasmid sequencing

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## Experimental Results

---

### 1. Validation the Feasibility of TRACE system-mediated directed evolution

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig1.png)

Figure 1. Feasibility validation of TRACE system.

#### (1) Construction of pTarget-EGFP

We synthesized the coding sequence of EGFP and obtained an EGFP fragment with homologous arms through PCR amplification. The pTarget plasmid (pCDH-CMV-T7) was subjected to double digestion with the restriction enzymes EcoR I and BamH I. Gel electrophoresis was conducted for both the digested fragments and the PCR products, followed by excision and purification of DNA bands corresponding to the desired sizes.
The purified DNA fragments were then ligated using homologous recombination (Gibson Assembly). The Gibson Assembly we used is a method of homologous recombination that involves the use of homologous arms. Homologous arms are short sequences added to the ends of the target DNA fragment, which have sequence overlaps with the recipient DNA. The Gibson Assembly method involves mixing the target DNA fragments, linearized vector DNA, and a recombinase enzyme system, and promoting the overlapping pairing between the homologous arms at iso-thermal conditions, resulting in the formation of ligated products. Specifically, the enzyme system includes an exonuclease, a polymerase, and a ligase. The exonuclease removes a small segment of bases from the 3' end of the target DNA fragment, generating a single-stranded overhang. Then, the polymerase uses this overhang as a primer to initiate the synthesis of a new DNA chain, connecting the target DNA fragment with the recipient DNA. Finally, the ligase joins the adjacent DNA chains of the ligation product to form a complete DNA construct.

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig2.png)

Figure 2. Construction of pTarget-EGFP plasmid.

Subsequently, the ligation product was transformed and plated onto agar plates containing ampicillin, followed by overnight incubation at 37°C. The following day, single colonies were selected and inoculated into an ampicillin-resistant LB medium for overnight cultivation. Plasmid extraction and sequencing were performed on the overnight cultures, resulting in the successful verification of the correct sequencing of the pTarget-EGFP plasmid (sequencing results specifically showed the bases near the site of the site-directed mutation, with the remaining sequences confirmed to be accurate).

#### (2) Construction of pTarget-BFP

Site-directed mutagenesis is a molecular biology technique used to introduce single nucleotide changes into DNA or RNA sequences selectively. This technique allows for the precise alteration of one or more nucleotides in a specific gene, resulting in changes to the target protein sequence. Site-directed mutagenesis is typically achieved by using a pair of primers that contain the desired changes in the nucleotides of interest for PCR amplification. During PCR, these primers anneal to complementary regions in the template DNA sequence and introduce the desired changes to the target nucleotide(s) during amplification. Through selective amplification and screening of individual colonies, DNA sequences with the desired mutations can be isolated from a mixed pool of DNA.
To obtain the pTarget-BFP plasmid, we used the pTarget-EGFP plasmid as a template and performed site-directed mutagenesis. Specifically, by using a pair of site-directed mutagenesis primers, we introduced a point mutation through PCR to change the tyrosine residue at position 67 of EGFP (green fluorescent protein) to histidine, resulting in the generation of BFP (blue fluorescent protein).

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig3.png)

Figure 3. Construction of pTarget-BFP plasmid.

#### (3) Transition from Blue to Green Fluorescence Protein: Validating the TRACE System

To validate the feasibility of the TRACE system, we introduced the pTarget-EBFP plasmid into HEK293FT cells that were pre-transfected with the pEditor plasmid to enable expression of EBFP. If the TRACE system was functional, upon activation of the TRACE system, we expected to observe a transition from blue fluorescence protein(BFP) to green fluorescence protein(GFP) due to the C-to-T mutation at position 199. The TRACE system was initiated by adding 1µM doxycycline, which induced the expression of AID-T7 RNAP-UGI to edit the EBFP gene downstream of the T7 promoter. We monitored the cells daily for the presence of green fluorescence. After 8 days of TRACE system activation, we successfully observed green fluorescence and recorded the images, indicating that the TRACE system was functioning properly.

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig4.png)

Figure 4. Conversion of BFP to GFP by the TRACE system.

### 2. Directed evolution of NAMPT gene by the TRACE systemevolution

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig5.png)

Figure 5. Search for highly active NAMPT mutants.

After verifying the feasibility of the TRACE system, we can utilize this system to evolve the key rate-limiting enzyme NAMPT involved in NAD+ biosynthesis. Before that, we require a reporting system for real-time monitoring of intracellular NAD+ concentration. Through literature search, we discovered that the team of Yuzheng Zhao from East China University of Science and Technology in Shanghai has developed the FiNad reporting system, which is specifically designed to monitor changes in NAD+ concentration both inside cells and in vivo. We obtained the ORF of FiNad from their published article, synthesized the fragment through DNA synthesis, and constructed it into the pCDH plasmid for the implementation of NAMPT-directed evolution experiments (With the help from team advisors).

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig6.png)

Figure 6. Construction of pCDH-CMV-mCherry-FiNad plasmid.

Due to the time constraints of the iGEM competition, we were unable to complete the entire process of NAMPT evolution experiments. However, through patent search, we discovered that some sites on the NAMPT protein have been confirmed through experiments such as prokaryotic expression, protein purification, and enzyme activity assays to enhance the enzymatic activity of NAMPT. We went forward and successfully constructed these mutated plasmids by our own, which can be used for subsequent enzyme activity validation.

#### (1) Construction of the wild-type NAMPT expressing plasmid

Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix. A ladder-like pattern of DNA fragmentation appeared upon agarose gel electrophoresis. Agarose gel electrophoresis is used to know how many different DNA fragments are present in a sample and how large they are relative to one another.
By comparing the bands in a sample to the DNA ladder using the gel documentation system, we can determine their approximate sizes. As a result, we confirmed that NAMPT fragments were successfully amplified, and pCDH vectors were also successfully linearized with restriction enzymes.

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig7.png)

Figure 7. NAMPT fragment and linearized vector preparation for molecular ligation.

We use the NanoDrop to quantify the amount of DNA sample.Nucleic acids absorb light at a wavelength of 260 nm. If a 260 nm light source shines on a sample, the amount of light that passes through the sample can be measured, and the amount of light absorbed by the sample can be inferred. For double-stranded DNA, an Optical Density (OD) of 1 at 260 nm correlates to a DNA concentration of 50 ng/μL so that DNA concentration can be easily calculated from OD measurements.

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig8.png)

Figure 8. NAMPT plasmid concentration measurement by Nanodrop.

#### (2) Site-directed mutagenesis of NAMPT genes

##### **Agarose Gel Electrophoresis**

Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix. A ladder-like pattern of DNA fragmentation appeared upon agarose gel electrophoresis. Agarose gel electrophoresis is used to know how many different DNA fragments are present in a sample and how large they are relative to one another.
Using the gel documentation system, it was confirmed that the NAMPT circular plasmid was successfully amplified.

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig9.png)

Figure 9. Site-directed mutagenesis of NAMPT genes.

##### **Pick multiple single clones for sequencing identification**

Bacteria grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.
As shown in the pictures below, the plates of bacteria cultured by different groups of students all grew monoclonal colonies.
Each student picked 3 individual bacterial colonies using sterile toothpicks, cultured them into a medium containing ampicillin antibiotic, and incubated them at 37°C overnight with shaking (300 rpm).

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig10.jpg)

Figure 10. The status of colony formation for each NAMPT mutant.

##### **Plasmid sequencing**

The monoclonal bacteria were amplified overnight and sent to the corresponding sequencing company for sequencing.The Sanger sequencing technique is the most commonly used method for determining DNA sequences of recombinant plasmids. Sanger sequencing involves the use of a DNA polymerase, a primer, unlabeled deoxynucleotide triphosphates (dNTPs), and fluorescently labeled dideoxynucleotide triphosphates (ddNTPs), where each base is labeled with a unique fluorophore. Incorporation of a ddNTP into the newly synthesized strand prevents the addition of subsequent nucleotides, stopping the further elongation of the DNA molecule and resulting in a DNA product with a fluorescently labeled ddNTP at the end of the strand. The sequence of nucleotides can then be determined by separating the DNA products by size.
By using SnapGene software, we could align DNA sequences with a reference sequence to verify mutagenesis. As shown in the pictures below, we successfully constructed five point mutations including D354E, G384C, L386P, V221A, and V221G.

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig11.png)

Figure 11. The sequence results for each NAMPT mutant.

### 3. [Future plan] Biochemical validation of highly active NAMPT mutants for NAD+ Bio-production

![](https://static.igem.wiki/teams/5005/wiki/ens/res/res-fig12.png)

Figure 12. Biochemical validation of highly active NAMPT mutants.

As long as we obtain the active NAMPT mutant(s), we will first validate the screen results through a series of biochemical assays including site-directed mutagenesis, prokaryotic expression, protein purification, and enzyme activity assays of NAMPT. If our mutant(s) passes these assays, we will go further and test its efficacy for NAD+ bio-production.

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