iGEM 2022 — Team uioslo-norway

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{% block title %}Notebook{% endblock %}
{% block lead %}by UiOslo{% endblock %}
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# Gene PCR

**Aim:** Extract and amplify our genomic UAP1 sequence from yeast DNA by Polymerase chain reaction (PCR).

**Protocol:** [Gene PCR](https://static.igem.wiki/teams/4450/wiki/protocol/pcr-amp-pur.pdf). We ran a 0.8% agarose gel electrophoresis to check our PCR product size.

**Conclusion:** We successfully amplified the UAP1 gene using PCR (Figure 1 showing a and of the size 1500bp). We can assemble our gene with a digested vector and transform it into chemically competent E. coli cells.

# Vector PCR

**Aim:** Linearizing and amplifying our vector IBA3 by Polymerase chain reaction (PCR).

**Protocol:** [Vector PCR](https://static.igem.wiki/teams/4450/wiki/protocol/pcr-amp-pur.pdf). We ran a 0.8% agarose gel electrophoresis to check our PCR product size.

**Conclusion:** We successfully amplified IBA3 vector using PCR (Figure 1 shows a band size 3215bp). We can now digest the vector with Dpn I enzyme.

**Result:**

![Isolation](https://static.igem.wiki/teams/4450/wiki/results/isolation.jpg)

**Figure 1:** To the left UAP1 1484bp, to the right IBA3-vector 3215bp.

# Dpn I digestion

**Aim:** : Linearization of the IBA3 plasmid for subsequent gene assembly.

**Protocol:** [Plasmid digestion](https://static.igem.wiki/teams/4450/wiki/protocol/dpni.pdf).

**Conclusion:** Ready plasmid for genomic assembly.

# Gene Assembly

**Aim:** Assemble our gene UAP1 with digested vector IBA3 and sequentially transform into competenc DHalpha E. coli cells.

**Protocol:** [Gene assembly](https://static.igem.wiki/teams/4450/wiki/protocol/assembly.pdf).

**Conclusion:** Ready plasmid with recombinant DNA for transformation.

# Bacteria Transformation and plating

**Aim:** Transform our cloned vector into the competent cells, DH-alpha E. coli, heat-shock and plate them on LB plate with ampicillin.

**Protocol:** [Transformation](https://static.igem.wiki/teams/4450/wiki/protocol/heat-shock-transformation.pdf).

![plate](https://static.igem.wiki/teams/4450/wiki/results/plate.png)

**Figure 2:** Three colonies and one tiny colony grew overnight on the LB plate.

**Conclusion:** Four colonies grew on the plate, however, have to verify the correct assembly of the cloned DNA construct by doing a colony PCR.

## Heat-shock

Mix the competent cells DH-alpha E. coli into the genes assembly and heat-shock them using thermocycler (-17 min).

# Inoculation

**Aim:** Pick the colonies that survived the ampicillin treatment and grow them in liquid cultures to amplify our plasmid DNA.

**Protocol:** [Batch of 5 mL E. Coli from agar plate](https://static.igem.wiki/teams/4450/wiki/protocol/e-coli.pdf).

**Result:** Turbid tubes which indicate bacterial growth.

**Conclusion:** Sufficient bacterial culture for subsequent experiments such as plasmid purification and protein induction.

## Inoculation of liquid culture

* Instead of the standard 5 mL/tube, we used 3 mL/tube
* Cultures were further subcultured from established tubes to 100 mL Erlenmeyer flasks with 20 mL/flask LB w/ampicillin (100 mg/ml)

# Colony PCR

**Aim:** To screen for genetically transformed and verify the assembly of the DNA construct.

**Protocol:** [Colony PCR](https://static.igem.wiki/teams/4450/wiki/protocol/pcr-amp-pur.pdf).

**Result:** Confirmation of expected size of amplicons.

![confirmation](https://static.igem.wiki/teams/4450/wiki/results/confirmation.jpg)

**Figure 3:** Colony PCR of colony 1-3, correct band size of approximately 1500bp confirmed in colony 1-3.

**Conclusion:** Assembly and transformation of the UAP1 gene into the IBA3 vector into DHalpha competent cells were successful in 3 of the 4 colonies.

# Miniprep

**Aim:** Isolate plasmid DNA from other DNA.

**Protocol:** [Miniprep](https://static.igem.wiki/teams/4450/wiki/protocol/miniprep.pdf).

**Result:** Sufficient amount of plasmid DNA in 3 cultures, determined by nanodrop.

| # | Concentration (ng/µL) |
| --- | --- |
| I1 | 199 |
| I2 | 164 |
| I3 | 99 |

**Conclusion:** Ready plasmid DNA for sequencing.

# Sequencing

**Aim:** Prove successful insert in vector.

**Protocol:** 6 tubes for 3 isolated plasmid samples were prepared. 3 tubes were prepared with 5 mM forward primer, and 3 with 5 mM reverse primer. 80-100 ng/µL plasmid DNA was added to each tube, one sample in both forward and reverse tube, then topped to 10 µL total in water. Sent for sanger sequencing to Eurofins Genomics.

**Result:** Successful insert of plasmid in all three cultures.

**Conclusion:** Undisputed proof that the target gene is inserted in the bacteria.

# Protein induction

**Aim:** Using AHTc to induce the expression of UPA1 gene.

**Protocol:** 10 mL bacterial culture is transferred to new flask, a dilution is prepared until 10 µL diluted AHTc is added to each.

**Result:** Colonies that produce the target protein.

**Conclusion:** Bacterial cultures in both induced and uninduced states are to be used for the analysis of protein to see if UAP1 is expressed as expected.

# Cell Lysis for nuclear protein extraction

**Aim:** Lyse the cells without using detergent to preserve the protein function.

**Protocol:** Pelleted with 3 000 rcf for 8 min. Lysis buffer added to 1/10 original volume (1 mL). Lysis buffer consistent with 200 mM, 0.5 mg/mL lysozyme, and 10 mM Tris. Bead lysis for ~40 seconds. Lysate separated into new tubes, then stored on ice for processing.

**Conclusion:** To determine the effectiveness of the cell lysis, functional assay and sds required.

# BSA assay, Bradford protein assay

**Aim:** Determining the amount of protein by calculation against BSA from induced and non-induced lysed bacterial cultures.

**Protocol:** [Bradford assay](https://static.igem.wiki/teams/4450/wiki/protocol/bsa.pdf).

**Result:**

![Bradford assay](https://static.igem.wiki/teams/4450/wiki/results/bradford.jpg)

**Figure 4:** from left to right, showing triplicates from induced (+)cultures: culture 1. A1-3, culture 2. B1-3, culture 3. C1-3, and culture 4. D1-3. Triplicates of non-induced (-) wells culture 1. A4-6, culture 2. B4-6, culture 3. C4-6, and culture 4. D, an4-6d enzyme (triplicates from A-D) well 7. Wall F-G shows triplicates of the BSA standard from blank 0 in well 1 to 2000 in well 7.

Estimated concentration using 5th degree polynomial regression:

| Induced (+; µg/µL) | Non-induced (-; µg/µL) |
| --- | --- |
| 1,368856 | 3,81238 |
| 1,528336 | 3,651112 |
| 1,164268 | 3,804776 |
| 0,479516 | 3,863736 |

**Conclusion:** Determined the concentration of the protein solutions in the different cultures.

# SDS-PAGE

**Aim:** Validate protein expression by the expected size.

**Protocol:** [SDS-PAGE](https://static.igem.wiki/teams/4450/wiki/protocol/sds-page.pdf).

**Result:** Confirmation of expected size of protein.

![SDS](https://static.igem.wiki/teams/4450/wiki/results/sds-1.jpg)

**Figure 5:** SDS-gel image with clear bands in induced 1, 2, 3, 5 and 6 of UAP1, a approximatly 53.5 kDa large protein.

**Conclusion:** Successful expression in induced vs non-induced.

# Dephosphorylation assay

**Aim:** Quantitative measurement of UAP1 activity.

**Protocol:** [Dephosphorylation assay](https://static.igem.wiki/teams/4450/wiki/protocol/dephosphorylation-assay.pdf).

**Result:**

![SDS](https://static.igem.wiki/teams/4450/wiki/results/assay-1.jpg)

**Figure 6:** A-C1-6 show a dilution of lysate from an uninduced culture batch, while D-F1-6 show a dilution of lysate from an induced culture batch. G-H1-3 are blank.

**Conclusion:** Unable to confirm increased activity caused by UAP1 in induced, versus non-induced culture.

[![University of Oslo](https://static.igem.wiki/teams/4450/wiki/sponsors/uio-logo.png)](https://www.uio.no/english/)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}by UiOslo{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4450/wiki/engin1.png)

### Introduction

Due to the complexity of biological systems, engineering it in any way is a hard, and often repetitive process of trial and error. In our project, we went through multiple cycles of design, engineering, and testing. From these steps, we obtained information, that went into rethinking and redesign of the project. On this page, we attempt to demonstrate our engineering cycle and out journey to success.

Summary of our troubleshooting and optimization of the following [parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D) to transform into *E. coli*.

* IDTDNA fragments for isothermal assembly that contain Bla promoter have been attempted to clone into pUC19 and transform using CaCl2 competent cells: Result: negative.
* Amplify genes from yeast gDNA with overhang that contains Bla promoter and restriction enzyme sequences (as appropriate) for ligation based assembly, attempt clone in pUC19: Result: negative.
* Amplify genes from yeast gDNA and use J23110 promoter on overhang as well as flanking sites for isothermal assembly, clone attempt into pUC19: Result: negative
* Amplify genes from yeast gDNA and use J23110 promoter on overhang as well as flanking sites for isothermal assembly, clone attempt into IBA3: Result: negative
* Prepare competent DH5a cells with [inoculation protocol](https://static.igem.wiki/teams/4450/wiki/protocol/preparation-of-chemically-competent-e-coli.pdf) to obtain better efficiency, hopefully, better yield (success rate of assemblies).
* Single attempts Amplify UAP1 gene with J23110 promoter and flanking sites for isothermal assembly, clone attempt into pUC19: Result: negative.
* Single attempts Amplify UAP1 gene with J23100 promoter and flanking sites for isothermal assembly, clone attempt into IBA3: Result: negative.
* Single attempts Amplify UAP1 gene with NO promoter (instead utilize the inducible pTet from IBA vector) and flanking sites for isothermal assembly, clone attempt into IBA3: Result: Successful.

![Engineering Cycle](https://static.igem.wiki/teams/4450/wiki/engineeringcolonies.svg)

![](https://static.igem.wiki/teams/4450/wiki/engin2.png)

### 1. Design

After months debate about a problem to solve, we identified the need for an easily obtainable, in vivo degradable and biocompatible material to use in medical applications. During brainstorming sessions, different options were presented as potential candidates from reviewing of pre-existing literature. From a paper published by Yadav et al. (2010), we were inspired by their idea to generate a material by fusing cellulose monomers with chitin elements, using the endogenous machinery of Komagataeibacter for cellulose production. We thought of improving this implementation by circumventing the need for amine sugars as a substrate thereby expanding upon the original work. Through the KEGG database, and literature three additional genes were selected in addition to two genes from the original publication. The design was changed multiple times throughout the project in response to problems in implementation or negative results. A more detailed explanation of this can be found in the [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4450/wiki/engin3.png)

### 2. Build

After the completion of the experimental design, we moved forward in the lab to bring the idea into reality. Initially we aimed at amplifying 3 genes from S. cerevisiae genomic DNA (gDNA) with the help of PCR. The primers were designed in away to allow for generation of parts that can be ligated together after restriction enzyme digestion. In addition to the above cloning approach we ordered parts from IDTDNA to be synthesized and sent to us. Our initial attempts were aiming to clone the necessary parts into certain cloning vector (pUC19) and the whole construct being under the control of the strong and constitutive promoter Bla. The attempts at amplifying the 3 individual genes from S. cerevisiae gDNA were successful. However, our ligation attempts either with restriction enzyme digestion approach or with isothermal assembly attempts were not successful. Eventually we opted for new designs utilizing a different promoter (J23110 from the iGEM Anderson promoter list) which led to the same results. Eventually we generated new designs, aiming at cloning the 3 necessary genes, plus one more ( GFA1? Convert fructose-6P to glucosamine-6P) and the target was a different expression vector (pASK-IBA3) under the control of the same constitutive promoter (J23110). After this attempt not yielding the desired results we decided to troubleshoot the cloning protocol, by isolating one gene (UAP1) and clone it directly in the pASK-IBA3 to verify that our cloning strategy is sound and at the same time demonstrate that the part in question, expresses properly using the pTet promoter from the pASKIBA3 vector which is an inducible promoter (turns on upon addition of anhydrotetracycline). Finally we successfully amplified UAP1 from yeast gDNA, cloned it into pASK-IBA3 and transformed it into E.coli where we attempted to express the protein. During a fast protein expression protocol we obtained crude protein lysate that we analysed with the help of SDS-PAGE and confirmed the expression of the gene of interest upon induction. At last we wanted to demonstrate the functionality of the protein expressed by using the crude cell lysate in a phosphatase assay, however we did not manage to finalize this aspect, although we have successfully used a colorimetric assay to detect phosphatase activity. This was necessary due to growth in recombinant organisms of the entire construct, which made us suspect that the changes were toxic to the bacteria. We went through two different designs, one using the original plasmid and process, mirroring the original publications method, and another attempt using a process recommended to us. We ended up transforming the single gene into our intermediate host, Escherichia coli, and proceeded to test this to validate insert and functional protein activity. More details on this process can be found in the [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) and [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4450/wiki/engin4.png)

### 3. Test

As part of the troubleshooting, we had to test the intermediate host thoroughly. Because of this, we tested both for proof of recombinant DNA insert with PCR amplification and digestion enzymes, and protein expression using SDS-PAGE and colorimetric assays. From this, we did not see the presence of recombinant DNA, but did see an increase in protein expression of the expected protein by size, and an increase in protein expression with increased metabolism of phosphates, the function of the protein encoded by recombinant DNA that we attempted to insert. With sequencing, we finally found that the plasmid did not contain our target DNA. A more detailed explanation of this process can be found in the [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D), [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) and [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

![](https://static.igem.wiki/teams/4450/wiki/engin5.png)

### 4. Learn

As explained above, this did not work as expected with all our experiments up-to this point. From this sets of trial and errors, we gained valuable knowledge. This tells us that the way we constructed and attempted to transform our bacteria was less than ideal and could even be toxic to our cell. And made us rethink the entire process; thus, a new engineering cycle began.

[![University of Oslo](https://static.igem.wiki/teams/4450/wiki/sponsors/uio-logo.png)](https://www.uio.no/english/)

[![Digital Life Norway](https://static.igem.wiki/teams/4450/wiki/dln-logo-eng.png)](https://www.digitallifenorway.org/)

[![Evogene](https://static.igem.wiki/teams/4450/wiki/sponsors/evogene.png)](https://www.mn.uio.no/ibv/english/research/sections/evogene/)

[![IDT](https://static.igem.wiki/teams/4450/wiki/sponsors/idt-logo-thumb-800.png)](https://eu.idtdna.com/)

[![novozymes](https://static.igem.wiki/teams/4450/wiki/sponsors/novozymes-a-s.jpg)](https://www.novozymes.com/en)

[![Oslo Mycology Group](https://static.igem.wiki/teams/4450/wiki/sponsors/omg-forslagtilfarger.png)](https://www.mn.uio.no/ibv/english/research/sections/evogene/groups/mycology/)

[![Empress Brewery](https://static.igem.wiki/teams/4450/wiki/sponsors/empressbrewery.png)](http://www.empressbrew.no/)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}by UiOslo{% endblock %}
{% block page\_content %}

## Research

During our design process, we figured out a problem we wanted to solve. From this we found a paper from Yadav, et al. (2011) [1]. This paper is a proposal for how to make a copolymer of cellulose and chitin in situ using cellulose producing bacteria cloned with genes from yeast. We choose to use *Saccharomyces cerevisiae* instead of *Candida albicans* as in the original research, and we wanted to expand the scope of production of this material.

To produce chitin monomers, its precursor UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a nucleotide sugar. N-acetylglucosamine (GlcNAc) is the monomer forming chitin and UDP-GlcNAc is the precursor to chitin. Chitin is the second most abundant polysaccharide after cellulose [1], a structurally similar polysaccharide. UDP-GlcNAc is synthesized by the hexosamine biosynthesis pathway (HBP), a pathway consistent of conserved enzymes, albeit mediated through different intermediate compounds in prokaryotes and eukaryotes, found in most living organisms [2, 3]. Despite the enzymes existing in most bacteria, taking part in synthesis of peptidoglycan, the expression is significantly lower than in yeasts, which depend on this pathway to produce chitin, a component of its cell wall [4].

The primary motivation to upregulate the expression of hexosamine biosynthesis enzymes is to produce a more biocompatible polymer then cellulose alone with specific applications in medicine and/or applications where high crystalline cellulose has a disadvantage as outlined more clearly by Yadav, et al and Helenius, et al [5, 6]. It has been proven that *Komagataeibacter* can incorporate amino-sugars in its endogenous cellulose synthase, creating a lysozyme-susceptible copolymer [7, 8].

![Overview of hexosamine biosynthesis pathway and upstream glycolysis in yeast.](https://static.igem.wiki/teams/4450/wiki/raimi-et-al-hexosamine-pathway-bba-k4450006.png)

**Figure 1:** Figure 1: Overview of hexosamine biosynthesis pathway and upstream glycolysis in yeast. Reused from a publication by Raimi, et al. (2020) in [Royal Society of Chemistry](https://royalsociety.org/journals/permissions/) licensed under [CC BY 3.0](https://creativecommons.org/licenses/by/3.0/) [9].

*Figure 1* is an overview of the hexosamine pathway. There are two entry points for substrate to enter biosynthesis UDP-GlcNAc. The first entry point is diffusion of a Glucosamine (GlcN) across the cell membrane from the extracellular space. This entry of UDP-GlcNAc biosynthesis by extracellular Glucosamine has been studied with limited success [7, 10]. This in turn limits the incorporation of non glucose (Glc) monomers by cellulose synthesis in *Komagataeibacter* as hypothesized and demonstrated by Yadav et al. [5]. They focused on this route of entry by expression of relevant enzymes from *Candida albicans* and observed an 18-fold increase in GlcNAc vs Glc monomers in their modified cellulose with the presence of extracellular GlcN. The other entry of substrate into UDP-GlcNAc biosynthesis is directly from glycolysis. Fructose-6-phosphate enters biosynthesis of UDP-GlcNAc by four different enzymes: GFA1, GNA1, PCM1/AGM1 and QRI1/UAP1. This is the pathway that we are modifying to improve the incorporation of GlcNAc vs Glc monomers by synthesis of UDP-GlcNAc from glucose directly. To increase the concentration of intracellular UDP-GlcNAc, genes related to hexosamine biosynthesis GFA1, GNA1, PCM1/AGM1 and QRI1/UAP1 are transformed into the mutant from *Saccharomyces cerevisiae*.

## Experiments

The source of our genetic material used as recombinant DNA is *Saccharomyces cerevisiae*. To extract the DNA, we designed primers and amplified the target genes from DNA extracted using lithium acetate to lyse the yeast. The DNA is further purified by gel extraction to a solution containing ideally only one gene. This material was then used in the transformation of our bacteria.

We have used the plasmid backbone pASG-IBA3 (IBA3), pUC19 and pBBR to clone our DNA sequences into our hosts. It was kindly provided to us by the Evogen laboratory. All plasmids are used in some way in our project, but to different purposes. IBA3 was as part of troubleshooting, because of bad experiences with pUC19 to assemble the DNA into out intermediate host *Escherichia coli*. This was necessary, as we didn’t know if it was the genetic construct or the design/method of insert that lead to poor results of cloning. pBBR is the plasmid used to insert the final construct from the intermediate host to transform *Komagataeibacter xylinus*. All plasmids are selected for in antibiotics containing medium, ampicillin for IBA3 and pUC19, and kanamycin for pBBR. Different transformation techniques were used in *E. coli* and *K. xylinus*, based on best practise found in literature. *E. coli* was transformed by preparation of competent cells in either calcium chloride or Inoue buffer and is transformed with heat-shock treatment. *K. xylinus* is prepared competent in HEPES buffer and transformed with electroporation.

When the bacteria were transformed, and growth on selective agarose plates were observed, the colonies were both suspended in a colony PCR and inoculated as a liquid culture in selective medium. From this, PCR of vector content using primers and gel electrophoresis is used to check if the insert is roughly the correct size. If this is the case, the liquid culture will be used for miniprep and lysis. The miniprep product is used for sequencing and lysate is used for protein analysis. The protein analysis consists of SDS-page to determine if protein is expressed by size, while a dephosphorylation assay is used to determine increased activity versus the parental strain.

## Protocols

Below you can read and download all the protocols we used for our project work.

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/assembly-img.png)

##### ASSEMBLY

We used New England BioLabs DNA ligation. Protocol largely...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/assembly.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/lysis-img.png)

##### BRADFORD ASSAY

This is the procedure for Bradford assay.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/bsa.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/lysis-img.png)

##### CELL LYSIS

This is the procedure for cell lysis.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/lysis.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/e-coli-img.png)

##### CULTURING *ESCHERICHIA COLI*

Escherichia coli (E. coli) is our intermediate host to proliferate the gene...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/e-coli.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/k-xylinus-img.png)

##### CULTURING *KOMAGATAEIBACTER XYLINUS*

This is the procedure for culturing *K. Xylinus*.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/k-xylinus.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/s-cerevisiae-img.png)

##### CULTURING *SACCHAROMYCES CEREVISIAE*

*Saccharomyces cerevisiae* (*S. cerevisiae*) is the source of our...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/s-cerevisiae.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/precipitation-img.png)

##### DNA PRECIPITATION

Nucleotide precipitation is a common technique for concentrating and...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/precipitation.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/dpni-img.png)

##### DPNI DIGESTION

Digestion and restriction cutting enzymes protocol. We use...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/dpni.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/gel-img.png)

##### GEL ELECTOPHORESIS AND EXTRACTION

This is the procedure for gel electophoresis and extraction.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/gel.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/heat-shock-transformation-img.png)

##### HEAT-SHOCK TRANSFORMATION

This is the procedure for heat-shock transformation.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/heat-shock-transformation.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/hs-img.png)

##### HESTRIN-SCHRAMM (HS) MEDIUM

Hestrin-Schramm medium is a common medium composition to...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/hs.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/lb-img.png)

##### LYSOGENY BROTH (LB) MEDIUM

Lysogeny broth (LB) is a nutritionally rich medium which is primarily...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/lb.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/miniprep-img.png)

##### MINIPREP

We used QIAprep Spin Miniprep Kit. This protocol is based on the official...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/miniprep.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/mb-img.png)

##### MISCELLANEOUS BUFFERS

This is the procedure for miscellaneous buffers.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/mb.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/pcr-amp-pur-img.png)

##### PCR AMPLIFICATION AND PURIFICATION

This is the procedure for PCR amplification and purification.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/pcr-amp-pur.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/pbs-img.png)

##### PHOSPHATE BUFFERED SALINE

Phosphate-buffered saline (PBS) is a buffer solution (pH ~7.4) commonly...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/pbs.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/dephosphorylation-assay-img.png)

##### DEPHOSPHORYLATION ASSAY

This is the procedure for dephosphorylation assay.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/dephosphorylation-assay.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/preparation-of-chemically-competent-e-coli-img.png)

##### PREPARATION OF CHEMICALLY COMPETENT *E. coli*

This is the procedure for preparation of chemically competent *E. coli*.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/preparation-of-chemically-competent-e-coli.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/preparation-of-chemically-supercompetent-e-coli-img.png)

##### PREPARATION OF CHEMICALLY SUPERCOMPETENT *E. coli*

This is the procedure for preparation of chemically supercompetent *E. coli*.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/preparation-of-chemically-supercompetent-e-coli.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/preparation-of-electrocompetent-k-xylinus-img.png)

##### PREPARATION OF ELECTROCOMPETENT *K. XYLINUS*

This protocol is a modified version of an existing protocol. To create...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/preparation-of-electrocompetent-k-xylinus.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/sds-page-img.png)

##### SDS-PAGE

This is the procedure for SDS-page.

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/sds-page.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/tb-img.png)

##### TRANSFORMATION BUFFERS

Different protocols for producing competent cells has over the years...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/tb.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/yeast-gdna-purification-img.png)

##### YEAST GENOMIC DNA EXTRACTION

Due to the rigidity of the yeast cell wall, we had to use a...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/yeast-gdna-purification.pdf)

![...](https://static.igem.wiki/teams/4450/wiki/protocol-img/ypd-img.png)

##### YEAST PEPTONE DEXTROSE (YPD) MEDIUM

Yeast peptone dextrose is a medium composition used to grow many common...

[Read more](https://static.igem.wiki/teams/4450/wiki/protocol/ypd.pdf)

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[## References](#hide-references)
[## References](#show-references)

1. Elieh-Ali-Komi, & Hamblin, M. R. (2016)

   Chitin and Chitosan: Production and Application of Versatile Biomedical Nanomaterials

   *International Journal of Advanced Research (Indore)*, 14(3): p.411-427

   [PMID: 27819009](https://pubmed.ncbi.nlm.nih.gov/27819009)
2. Lockhart DEA, Stanley M, Raimi OG, Robinson DA, Boldovjakova D, Squair DR, Ferenbach AT, Fang W, van Aalten DMF (2020)

   Targeting a critical step in fungal hexosamine biosynthesis

   *J Biol Chem.*, 295(26): p. 8678-8691

   [DOI: 10.1074/jbc.RA120.012985](https://doi.org/10.1074/jbc.ra120.012985)
3. Li S, Kang J, Yu W, Zhou Y, Zhang W, Xin Y, Ma Y (2012)

   Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG\_1556 and essentiality of M. smegmatis MSMEG\_1556

   *PLoS One*, 7(8): e42769

   [DOI: 10.1371/journal.pone.0042769](https://doi.org/10.1371/journal.pone.0042769)
4. Brown HE, Esher SK, Alspaugh JA (2020)

   Chitin: A "Hidden Figure" in the Fungal Cell Wall

   *Curr Top Microbiol Immunol.*, 425: 83-111

   [DOI: 10.1007/82\_2019\_184](https://doi.org/10.1007/82_2019_184)
5. Yadav V, Paniliatis BJ, Shi H, Lee K, Cebe P, Kaplan DL (2010)

   Novel in vivo-degradable cellulose-chitin copolymer from metabolically engineered Gluconacetobacter xylinus

   *Appl Environ Microbiol.*, 76(18): 6257-65

   [DOI: 10.1128/AEM.00698-10](https://doi.org/10.1128/aem.00698-10)
6. Helenius G, Bäckdahl H, Bodin A, Nannmark U, Gatenholm P, Risberg B (2006)

   In vivo biocompatibility of bacterial cellulose

   *J Biomed Mater Res A.*, 76(2): 431-8

   [DOI: 10.1002/jbm.a.30570](https://doi.org/10.1002/jbm.a.30570)
7. Shirai A, Takahashi M, Kaneko H, Nishimura S, Ogawa M, Nishi N, Tokura S (1994)

   Biosynthesis of a novel polysaccharide by Acetobacter xylinum

   *Int J Biol Macromol*, 16(6): 297-300

   [DOI: 10.1016/0141-8130(94)90059-0](https://doi.org/10.1016/0141-8130%2894%2990059-0)
8. Ogawa R, Miura Y, Tokura S, Koriyama T (1992)

   Susceptibilities of bacterial cellulose containing N-acetylglucosamine residues for cellulolytic and chitinolytic enzymes

   *Int J Biol Macromol.*, 14(6): 343-7

   [DOI: 10.1016/s0141-8130(05)80076-5](https://doi.org/10.1016/s0141-8130%2805%2980076-5)
9. Raimi, Olawale G. and Hurtado-Guerrero, Ramon and Borodkin, Vladimir and Ferenbach, Andrew and Urbaniak, Michael D. and Ferguson, Michael A. J. and van Aalten, Daan M. F. (2020)

   A mechanism-inspired UDP-N-acetylglucosamine pyrophosphorylase inhibitor

   *RSC Chem. Biol.*, 1: p. 13-25

   [DOI: 10.1039/C9CB00017H](http://dx.doi.org/10.1039/C9CB00017H)
10. Lee JW, Deng F, Yeomans WG, Allen AL, Gross RA, Kaplan DL (2001)

    Direct incorporation of glucosamine and N-acetylglucosamine into exopolymers by Gluconacetobacter xylinus (=Acetobacter xylinum) ATCC 10245: production of chitosan-cellulose and chitin-cellulose exopolymers

    *Appl Environ Microbiol.*, 67(9): 3970-5

    [PMID: 11525993](https://pubmed.ncbi.nlm.nih.gov/11525993)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}by UiOslo{% endblock %}
{% block page\_content %}

# Gene isolation

From *Saccharomyces cerevisiae* UAP1/QRI1 is isolated. This is done by genomic DNA, using primers to amplify the gene of interest. The final amplicon of UAP1/QRI1 is 1 434 bp as observed in figure 1. Furthermore, we amplified the plasmid backbone used in testing pASG-IBA3 (IBA3) from *Escherichia coli* (E. coli) DH5α using primers. The final amplicon of this is 3 115 bp as observed from figure 1.

![PCR product](https://static.igem.wiki/teams/4450/wiki/results/isolation.jpg)

**Figure 1:** Gel image of 4 µL PCR product of the target gene UAP1/QRI1 and vector pASG-IBA3 with 1 µL DNA loading buffer.

# PCR amplification of vector insert

After successful transformation confirmed by growth on ampicillin agar plates, single colonies were picked and amplified using primers specific to the cloning site of IBA3. From these four clones, we expected a band around 1 500 bp to indicate successful insert of UAP1/QRI1. As expected, we did get amplified bands around 1 500 bp.

![PCR product](https://static.igem.wiki/teams/4450/wiki/results/confirmation.jpg)

**Figure 2:** Gel image of 4 µL PCR product of the target gene UAP1/QRI1 with 1 µL DNA loading buffer.

# Bradford assay

We quantified protein content using Bradford assay. From what we see in Figure 3:

![PCR product](https://static.igem.wiki/teams/4450/wiki/results/bradford.jpg)

**Figure 3:** Plate overview after incubation of plate with known concentration standard at the bottom from 0 to 2 µg/mL. Top show protein concentration from induced cultures (+) and non-induced cultures (-). A vehicle control (E) was also added, only loaded with the lysis buffer to account for the lysozyme concentration.

From this, protein concentration is estimated by 5-degree polynomial regression of the standard, then unknown protein concentration is calculated and shown in Table 1.

| # | Induced (+; µg/µL) | Non-induced (-; µg/µL) |
| --- | --- | --- |
| 1 | 1,368856 | 3,81238 |
| 2 | 1,528336 | 3,651112 |
| 3 | 1,164268 | 3,804776 |
| 4 | 0,479516 | 3,863736 |

**Table 1:** Calculated concentration of protein from Bradford assay using the recommended.

# Sequencing

From sequencing of the entire pASG-IBA3 plasmid, we got the following sequence as a vector map.

![PCR product](https://static.igem.wiki/teams/4450/wiki/results/uap1-iba.png)

**Figure 4:** Vector map representation of the sequence result with annotations.

Confirmation of gene sequence was done using alignment with NCBI alignment of two or more sequences with standard settings for nucleotide-nucleotide BLAST. The sequenced file was compared with the known gene (sequence in part, [BBa\_K4450002](http://parts.igem.org/Part%3ABBa_K4450002)). Alignment is found here:

|  |
| --- |
| [Sequence 1](https://static.igem.wiki/teams/4450/wiki/results/sequence-1.pdf) |
| [Sequence 2](https://static.igem.wiki/teams/4450/wiki/results/sequence-2.pdf) |
| [sequence.json](https://static.igem.wiki/teams/4450/wiki/results/sequence.json) |

# SDS-PAGE

To check for protein expression, we decided to run an SDS-page. Six cultures were prepared with the mutation, separated in induced and non-induced batches. From this, we got the following image which show clear expression in the induced cultures versus the non-induced cells. The protein is approximately 53.5 kDa large. Only batch 4 showed no expression of the protein.

![Gel image](https://static.igem.wiki/teams/4450/wiki/results/sds-1.jpg)

**Figure 5:** Gel image of SDS-page loaded with 6 batches of transformed bacteria.

# Dephosphorylation assay

From the dephosphorylation assay we found the following after incubation of plate.

![assay](https://static.igem.wiki/teams/4450/wiki/results/assay-1.jpg)

**Figure 6:** Wells A-C1-6 show a dilution of lysate from an uninduced culture batch, while D-F1-6 show a dilution of lysate from an induced culture batch. G-H1-3 are blank.

This plate was measured at 655 nm, and we found the following absorbance values.

![assay](https://static.igem.wiki/teams/4450/wiki/results/assay-2.png)

**Figure 7: Absorbance measurements at A655nm**.

No statistical observable effect between induced and uninduced culture lysate. This might suggest low expression of UAP1 relative to other phosphatases expressed in the lysate. Another explanation could be denaturation of protein, inhibiting protein activity due to method of cell lysis.

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{% endblock %}