iGEM 2021 — Team KCIS_NewTaipei

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# Team:KCIS NewTaipei/Notebook

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  PROJECT
  + [DESCRIPTION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Description)
  + [IMPLEMENTATION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Implementation)
  + [NOTEBOOK](https://2021.igem.org/Team%3AKCIS_NewTaipei/Notebook)
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  WET LAB
  + [EXPERIMENTS](https://2021.igem.org/Team%3AKCIS_NewTaipei/Experiments)
  + [PARTS](https://2021.igem.org/Team%3AKCIS_NewTaipei/Parts)
  + [PROOF OF CONCEPT](https://2021.igem.org/Team%3AKCIS_NewTaipei/Proof_Of_Concept)
  + [ENGINEERING](https://2021.igem.org/Team%3AKCIS_NewTaipei/Engineering)
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  DRY LAB
  + [MODEL](https://2021.igem.org/Team%3AKCIS_NewTaipei/Model)
  + [HARDWARE](https://2021.igem.org/Team%3AKCIS_NewTaipei/Hardware)
  + [SOFTWARE](https://2021.igem.org/Team%3AKCIS_NewTaipei/Software)
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  HUMAN

  PRACTICES
  + [COLLABORATIONS](https://2021.igem.org/Team%3AKCIS_NewTaipei/Collaborations)
  + [EDUCATION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Education)
  + [ENTREPRENEURSHIP](https://2021.igem.org/Team%3AKCIS_NewTaipei/Entrepreneurship)
  + [HUMAN PRACTICE](https://2021.igem.org/Team%3AKCIS_NewTaipei/Human_Practices)
  + [CONTRIBUTION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Contribution)
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  TEAM
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Notebook

NOTEBOOK
*NOTEBOOK*

* [LAB

  NOTEBOOK](#lnLink)
* [DAILY

  UPDATE](#duLink)

Lab Notebook

This notebook is used for recording the progress of our experiments.

Daily Update

We planned our project schedule with this table.

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# Team:KCIS NewTaipei/Engineering

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  PROJECT
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  + [NOTEBOOK](https://2021.igem.org/Team%3AKCIS_NewTaipei/Notebook)
  + [SAFETY](https://2021.igem.org/Team%3AKCIS_NewTaipei/Safety)
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  WET LAB
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  + [PARTS](https://2021.igem.org/Team%3AKCIS_NewTaipei/Parts)
  + [PROOF OF CONCEPT](https://2021.igem.org/Team%3AKCIS_NewTaipei/Proof_Of_Concept)
  + [ENGINEERING](https://2021.igem.org/Team%3AKCIS_NewTaipei/Engineering)
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  DRY LAB
  + [MODEL](https://2021.igem.org/Team%3AKCIS_NewTaipei/Model)
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  HUMAN

  PRACTICES
  + [COLLABORATIONS](https://2021.igem.org/Team%3AKCIS_NewTaipei/Collaborations)
  + [EDUCATION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Education)
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  TEAM
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Implementation

ENGINEERING
*ENGINEERING*

* [SUMMARY](#SummaLink)
* [HBD PREPARATION](#HbdLink)
* [CLONING CYCLE](#cloneLink)
* [COLONY PCR](#colonyLink)
* [IMPROVEMENT](#improveLink)
* [A NEW CLONING

  CYCLE](#cycleLink)
* [COLONY PCR](#colonyILink)
* [SEQUENCING](#sequencingLink)
* [CONCLUSION](#ConcluLink)

Summary

We inserted our gene of interest, HBD, into bacterial cells through cloning and proved the engineering success through colony polymerase chain reaction and sequencing.

HBD Preparation

Using the TM (Melting temperature) calculator, we predicted the annealing temperature of the primer (66°C). However, since we are amplifying HBD directly from EcN (E-Coli Nissle 1917), a biologically complex system, we worried that if the predicted annealing temperature is higher than the actual value, primers would not be able to anneal properly. Hence, gradient PCR, with annealing temperature 66°C , 64°C, 61°C, and 57°C, was utilized to determine the optimal annealing temperature of our primer (Figure 1).

![](https://static.igem.org/mediawiki/2021/9/94/T--KCIS_NewTaipei--ENGPic2.png)

Figure 1. Gradient PCR to determine optimal annealing temperature. The annealing temperature from the leftmost lane to the rightmost lane was set to be 66°C , 64°C, 61°C, and 57°C, respectively. Bands at 888 bp appeared in all four lanes. The letter M is used to indicate the lane loaded with a 1kb DNA ladder.

Gel electrophoresis (Figure 1) confirms the presence of the HBD gene, since bands at 888 bp appeared in all samples. The result proves that 66℃ is the optimal annealing temperature since the band size at 888 bp is the largest for the lane corresponding to 66℃. We thus purified that band and digested it overnight. The concentration of HBD gene after digestion was measured to be 38ng/μL.

Cloning Cycle

We performed mini-prep to isolate the pET28a plasmid from the bacterial cell, washing and purifying the vector with Zymo DNA isolation kit. The plasmids pET28a were then digested overnight. The concentration of the vector after digestion was measured to be 11ng/μL.

Digested pET28a and HBD was then ligated for 2 hours. 0.8 μL of digested HBD and 4.5 μL of digested pET28a were added in the experimental group, while 4.5 μL of digested pET28a were added in the control group. During ligation, the segments, the HBD gene and the pET28a vector, were expected to be ligated by ligase at the restriction enzyme cut sites (EcoR1 & Xho1). The ligated plasmid was transformed into DH5α (competent E. coli cells) , which were then cultured overnight (Figure 2).

![](https://static.igem.org/mediawiki/2021/5/5a/T--KCIS_NewTaipei--ENGPic4.png)

Figure 2. Overnight cultures. For both the negative control and experimental group, we plated the bacteria with 50 and 200μL of sample.

The result is inconsistent with our expectations. Normally, we would expect there to be no colonies on the negative control plate since the negative control strain does not carry resistance. On our negative control plate, however, a significant amount of bacteria grew. We still decided to select bacterial strains for colony PCR because we presumed that the colonies on the agar plate all contain pET28a plasmid inserted with the HBD gene.

Colony PCR

We perform colony PCR on 10 randomly chosen colonies from the 200 μL pET28a-HBD plate. We ran gel electrophoresis using these 10 samples (Figure 3).

![](https://static.igem.org/mediawiki/2021/2/2a/T--KCIS_NewTaipei--ENGPic5.png)

Figure 3. Result of colony PCR. The letter M is used to indicate the lane loaded with DNA 1 kb ladder.

According to figure 3, the target gene construct (HBD gene in the T7 system), which is 977 bp, did not appear in the region between the 900 bp and 1000 bp mark as expected.

Error Analysis

We believe the error was due to not cutting the plasmid pET28a thoroughly during digestion. This would result in some of the plasmid not ligating with the HBD gene, undergoing self-ligation. To resolve this problem, we thus decided to amplify the plasmid backbone directly via PCR, forcing it to stay in linear form by separating two segments of circular DNA, preventing self ligation. We believed that this could significantly increase the success rate.

New Cloning Cycle

In our new trial, we used the HBD gene from the first trial. The plasmid backbone pET28a, on the other hand, was amplified directly using PCR. Gel electrophoresis, which presented the band at 5369 bp, confirmed the presence of pET28a backbone (Figure 4).

![](https://static.igem.org/mediawiki/2021/2/24/T--KCIS_NewTaipei--ENGPic6.png)

Figure 4. Gel electrophoresis confirms the presence of pET28a. The letter M is used to indicate the lane loaded with DNA 1 kb ladder.

We ligated HBD with pET28a and transformed it into DH5𝛼. The bacterial cells were then cultured overnight (Figure 5).

![](https://static.igem.org/mediawiki/2021/3/3c/T--KCIS_NewTaipei--ENGPic7.png)

Figure 5. The agar plates culturing bacteria containing linear vectors-only did not show any colonies, while colonies of the bacteria containing the pET28a plasmid inserted with the HBD gene successfully grew on the plates. The culturing results confirmed our expectation.

Colony PCR

We again performed colony PCR on 10 randomly chosen colonies from the 200 μL pET28a-HBD plate. The gel electrophoresis result, which presented the band at 977 bp, confirmed the presence of the inserted HBD gene in the pET28a plasmid construct.

![](https://static.igem.org/mediawiki/2021/d/d7/T--KCIS_NewTaipei--ENGPic8.png)

Figure 6. Result of colony PCR. The letter M is used to indicate the lane loaded with DNA 1 kb ladder.

Sequencing

To ensure that the resulting DNA sequence from PCR matches our designed gene of interest, we sent our construct, pET28a-HBD, to sequencing. The result matches the expected sequence (Figure 7)

![](https://static.igem.org/mediawiki/2021/7/7b/T--KCIS_NewTaipei--ENGPic9.png)

Figure 7. The result of sequencing demonstrates a matching rate of 98% out of 1024 bp.

Conclusion

The results of colony PCR and sequencing both demonstrated the success of our cloning cycle. We, therefore, can move on to further steps of our project which consists of confirming the expression of HBD protein through SDS-PAGE.

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# Team:KCIS NewTaipei/Experiments

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  PROJECT
  + [DESCRIPTION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Description)
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  + [NOTEBOOK](https://2021.igem.org/Team%3AKCIS_NewTaipei/Notebook)
  + [SAFETY](https://2021.igem.org/Team%3AKCIS_NewTaipei/Safety)
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  WET LAB
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  + [PARTS](https://2021.igem.org/Team%3AKCIS_NewTaipei/Parts)
  + [PROOF OF CONCEPT](https://2021.igem.org/Team%3AKCIS_NewTaipei/Proof_Of_Concept)
  + [ENGINEERING](https://2021.igem.org/Team%3AKCIS_NewTaipei/Engineering)
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  DRY LAB
  + [MODEL](https://2021.igem.org/Team%3AKCIS_NewTaipei/Model)
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  + [SOFTWARE](https://2021.igem.org/Team%3AKCIS_NewTaipei/Software)
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  HUMAN

  PRACTICES
  + [COLLABORATIONS](https://2021.igem.org/Team%3AKCIS_NewTaipei/Collaborations)
  + [EDUCATION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Education)
  + [ENTREPRENEURSHIP](https://2021.igem.org/Team%3AKCIS_NewTaipei/Entrepreneurship)
  + [HUMAN PRACTICE](https://2021.igem.org/Team%3AKCIS_NewTaipei/Human_Practices)
  + [CONTRIBUTION](https://2021.igem.org/Team%3AKCIS_NewTaipei/Contribution)
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  TEAM
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Experiment

EXPERIMENT
*EXPERIMENT*

* [PARTS

  DESCRIPTION](#pdLink)
* [CONFIRMATION

  OF HBD & BCD

  OVEREXPRESSION](#confLink)
* [ASSAYS OF

  HBD & BCD

  ACTIVITY](#assayLink)
* [UPREGULATION

  OF VDR INDUCED

  BY BUTYRATE](#uprLink)

Due to COVID-19, we couldn’t finish our wet lab since we only had one month to do the experiment. Some of the future experiments we planned can be performed if our team decides to continue on phase 2 in the next iGEM cycle.

Parts Description

![](https://static.igem.org/mediawiki/2021/6/60/T--KCIS_NewTaipei--exp_Figure1.png)

\*Fig.1 Gene construct for HBD and BCD gene

Our HBD was not synthesized as a composite part, therefore it was obtained directly from E.coli Nissle
1917 strain (EcN). To construct the HBD expression plasmid, primers possessing the restriction enzyme
sites EcoRI and XhoI were used to amplify the HBD gene via PCR from EcN genomic DNA. The HBD gene was
inserted into pET28a.

On the other hand, the BCD gene, as a composite part, was designed using Benchling. The BCD gene
sequence was obtained from the genomic DNA of Faecalibacterium prausnitzii. The BCD gene, flanked by
restriction sites EcoRI and SpeI, was synthesized by Twist Bioscience and was inserted into pSB1C3 via
BioBrick Assembly.

![](https://static.igem.org/mediawiki/2021/b/ba/T--KCIS_NewTaipei--exp_Figure2.png)

\*Fig.2 Insertion of our gene of interest, HBD gene and BCD gene, into our vector, pET28a and pSB1C3

We then transformed engineered pET28a and pSB1C3 into E.coli DH5𝛼 competent cell separately. The
bacterial strains were cultured in Luria Broth medium, including kanamycin for pET28a, chloramphenicol
for pSB1C3. After using colony PCR to test the success of transformation, the plasmids were extracted
using Miniprep and inserted into BLR(DE3) competent cells since DH5𝛼 does not acquire the T7 promoter
system. The culture was subsequently left to grow overnight in LB medium for protein expression assay.

![](https://static.igem.org/mediawiki/2021/2/25/T--KCIS_NewTaipei--exp_Figure3.png)

\*Fig.3 Inserting HBD and BCD into competent cells.

Confirmation of HBD & BCD
 Overexpression

Since pET28a is a lac operon inducible system, we added IPTG into our bacterial culture to induce protein
expression. We set the IPTG induced culture as the experimental group, while uninduced and culture as
negative control; the wildtype culture was included in the negative control group to prevent leaky
expression from the lac operon. For the BCD construct, since lac operon was not present, we set the
engineered culture as the positive control, while wildtype culture as negative control.

We lysed our culture by heating the sample. Both HBD and BCD protein were purified using nickel column
chromatography. The concentration of the protein was determined using a spectrophotometer at a wavelength of
280 nm.

Assays of HBD and BCD Activity

HBD

The HBD protein catalyzes the formation of 3-hydroxybutyryl-CoA from acetyl-CoA, while oxidizing NADH to
NAD+.

![](https://static.igem.org/mediawiki/2021/f/fd/T--KCIS_NewTaipei--exp_Figure4.png)

\*Fig.4 Reaction catalyzed by HBD

We added a fixed amount of acetyl-CoA and NADH, while varying the concentration of purified HBD protein. The
activity of the protein was accessed through measuring the concentration of the reactant NADH, with a
spectrophotometer at a wavelength of 340 nm.

BCD

We implemented the FadB-coupled assay proposed by Park et al. The BCD catalysis reaction was coupled with
that of FadB, which catalyzes the formation of acetoacetyl-CoA to crotonyl-CoA, with simultaneous reduction
of NAD+ to NADH.

![](https://static.igem.org/mediawiki/2021/3/39/T--KCIS_NewTaipei--exp_Figure5.png)

\*Fig.5 Reaction catalyzed by BCD

We added a fixed amount of butyric acid, acetyl-CoA, FAD, and NAD+, while varying the concentration of the
purified BCD protein, for the reverse pathway to proceed. The activity of the protein was again accessed
through measuring the concentration of the product NADH using the same method mentioned above.

Upregulation of VDR Induced by
 Butyrate

In order to confirm that butyrate is able to upregulate VDR, we treated HCT cell lines with various dosages
of butyrate to quantify VDR protein expression. Sodium butyrate was dissolved in PBS. The dosage of butyrate
ranged from 0mM (PBS only) to 3mM, with a constant leap of 0.5mM. The HCT cell lines were cultured in DMEM.
The medium of culture was replaced every day.

![](https://static.igem.org/mediawiki/2021/e/e0/T--KCIS_NewTaipei--exp_Figure6.png)

\*Fig.6 Adding different doses of sodium butyrate into HCT cell lines.

We left HCT cells to grow in a medium containing butyrate for 48 hours. The cells were then harvested,
washed in ice-cold PBS, and lysed by heating. Protease inhibitors were added to prevent protein degradation.
The lysate was then sonicated, centrifuged, and quantified using Bio-Rad protein assay.

After quantification, the lysate was again analyzed using SDS-PAGE. The band at 48kDa was identified and
quantified using Bio-Rad Image software. The experiment procedure was repeated three times. The datas were
used for modeling efforts.

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# Team:KCIS NewTaipei/Results

# Results

You can describe the results of your project and your future plans here.

### 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](http://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. 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.

### Inspiration

See how other teams presented their results.

* [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)