Difference between revisions of "Part:BBa K4719013"

Latest revision as of 14:39, 12 October 2023

AGM1-NAG5-UAP1 operon for incorporation of N-acetylglucosamine into bacterial cellulose

Sequence and Features

Assembly Compatibility:

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Illegal EcoRI site found at 3794
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Illegal SpeI site found at 4620
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Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for in vivo alterations of Komagataeibacter xylinus bacterial cellulose polymer composition. Firstly, we chose to produce a cellulose-chitin copolymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.

This specific composite part was used for bacterial cellulose-chitin polymer production. It was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. We employed two strategies to produce this material:
1. The first approach was to add N-acetylglucosamine into the growth medium BBa_K4719013.
2. Second one was the production of N-acetylglucosamine by K. xylinus from common sugars such as glucose, fructose, and sucrose in the growth medium BBa_K4719014.

Usage and Biology

This construct produces an excess of UDP-N-acetylglucosamine (GlcNAc) in K. xylinus, which can be used by cellulose synthase to produce bacterial cellulose-chitin polymer. Incorporation of N-acetylglucosamine into cellulose has been previously demonstrated by M. H. Tan (2019) and V. Yadav (2010). K. xylinus requires genetic modification to incorporate GlcNAc into the bacterial cellulose as the natural conversion to UDP-GlcNAc is inefficient. The genes in this composite code for proteins that are responsible for the uptake of extracellular GlcNAc and conversion to UDP-GlcNAc. Therefore, the growth medium has to be supplemented with GlcNAc.
Bacterial cellulose-chitin copolymer has applications in the biomedicine field due to in vivo biodegradability by the lysozyme. However, this copolymer is an intermediate substance in our system since we chose to improve the qualities of the material by deacetylation. This second enzymatic step produces a cellulose-chitosan copolymer with the added benefits of antibacterial activity and primary amino groups for easier functionalization.
Since polymer production occurs in K. xylinus, it requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable plasmid backbone for Komagateibacter species. We performed PCR of the plasmid eliminating mRFP in order to preserve Anderson promoter J23104 (BBa_J23104), RBS (BBa_B0034) and terminator ( BBa_B0015). AGM1, NAG5 and UAP1 genes were assembled into the backbone by Gibson assembly.

Experimental characterisation

Polymer production

Bacterial cellulose-chitin polymer is synthesized by K. xylinus grown in glucose yeast extract broth (GYB) while shaking at 180 rpm at 28°C for 7 days. Varying concentrations of glucose and N-acetylglucosamine have been added to determine the best combination of growth conditions. We experimentally determined that the optimal carbon source ratio is 1 % glucose and 1 % N-acetylglucosamine for the highest incorporation of chitin monomers into bacterial cellulose.

**Figure 1. Bacterial cellulose-chitin copolymer. A** - bacterial cellulose control group, grown on 2 % glucose. B - bacterial cellulose-chitin copolymer, grown on 1 % glucose and 1 % N-acetylglucosamine. The morphology of copolymer is different from natural bacterial cellulose, with a looser association of pellicles. This is consistent with previous work done by V. Yadav et al. (2010).

Gene expression analysis by RT-qPCR

In order to verify synthetic operons AGM1-NAG5-UAP1 expression in K. xylinus, we performed real-time quantitative PCR. We were unable to conduct a quantitative analysis of the results due to a shortage of time for generating biological replicates. But, we could compare cycle threshold (Ct) values. This measurement shows how many PCR cycles are required to obtain positive results (Bonacorsi et al., 2021). Expression of every selected gene was detectable because all Ct values were lower than 37.

**Figure 2. RT-qPCR Ct values for *AGM1-NAG5-UAP1*.** As expected, Ct value for the housekeeping gene of DNA gyrase subunit B (*gyrB*) is lower compared to the operon, indicating a higher expression. When comparing expression between genes in *AGM1-NAG5-UAP1*, we noticed that Ct values are very similar for all three genes, meaning that the whole operon is transcribed.

FTIR spectra of bacterial cellulose-chitin copolymer

The adsorption band at 1589 cm^-1 closely corresponds with OH deformation vibration as described in the literature [3]. This vibration could be attributed to 2’ OH and is the most apparent in the A curve of bacterial cellulose polymer control group. The absorption intensity of this peak decreases with the incorporation of N-acetylgluscosamine. The second band at 1651 cm^-1 is amine I vibration, confirming the incorporation of N-acetylglucosamine [4]. However, the wavenumbers of bands of OH deformation vibration and amine I vibration fall into the same region, therefore, the 1651 cm^-1 band in the control group of bacterial cellulose is visible. With this in mind, we can compare the composition of polymers by observing how both bands change respectively.
Since FTIR is a qualitative measure, both B and C curves show that the polymer composition has changed from natural bacterial cellulose to bacterial cellulose-chitin copolymer.

Fluorescence analysis of N-acetylglucosamine incorporation

To determine if the polymer contains N-acetylglucosamine residues, we used fluorescent lectin staining with wheat germ agglutinin (WGA), Alexa Fluor™ 680 conjugate selectively binding to N-acetylglucosamine. Therefore, natural cellulose should not give a visible signal at this wavelength.
Construct for incorporation of N-acetylglucosamine into bacterial cellulose improved fluorescence signal significantly, showing consistent distribution of the target monomers.

**Figure 4. Fluorescent lectin staining with WGA. A** - bacterial cellulose grown on 2 % glucose (control group). B - bacterial cellulose control group grown on 1 % glucose and 1 % N-acetylglucosamine. C - bacterial cellulose-chitin copolymer grown on 1 % glucose and 1 % N-acetylglucosamine. Fluorescence signal intensity corresponds with the amount of N-acetylglucosamine in the polymer, therefore unmodified *K. xylinus* is capable of slight incorporation of N-acetylglucosamine. However, **our composite part improves this process significantly**.

Chracterization of polymer surface with SEM

For further verification of bacterial cellulose-chitin polymer structural differences from natural bacterial cellulose, we performed scanning electron microscopy (SEM).

**Figure 5. Bacterial cellulose-chitin copolymer surface characterization by SEM. A** - bacterial cellulose grown on 2 % glucose (control group). B - bacterial cellulose-chitin copolymer grown on 1 % glucose and 1 % N-acetylglucosamine. The arrangement of fibers is much finer in the copolymer due to altered hydrogen bonding properties between glucose monomers. The crystallinity decreases as the fibers of the polymer do not have such strong intramolecular bonds.

Growth burden

In order to work with E. coli for designing and testing constructs, the growth burden of said synthetic biology tools has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified E. coli DH5α. The composite AGM1,NAG5,UAP1 did not inhibit the growth of E. coli as seen in Figure 6.

References

1. Teh, M.Y. et al. (2019) ‘An expanded synthetic biology toolkit for gene expression control in acetobacteraceae’, ACS Synthetic Biology, 8(4), pp. 708–723. doi:10.1021/acssynbio.8b00168.
2. Yadav, V. et al. (2010) ‘Novel in vivo degradable cellulose-chitin copolymer from metabolically engineered gluconacetobacter xylinus’, Applied and Environmental Microbiology, 76(18), pp. 6257–6265. doi:10.1128/aem.00698-10.
3. Socrates, G. Infrared and Raman characteristic group frequencies: tables and charts. John Wiley and Sons, LTD. - 3rd ed., 2002
4. Barth, A. (2007) ‘Infrared spectroscopy of proteins’, Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1767(9), pp. 1073–1101. doi:10.1016/j.bbabio.2007.06.004.
5. Bonacorsi, S. et al. (2021) ‘Systematic review on the correlation of quantitative PCR cycle threshold values of gastrointestinal pathogens with patient clinical presentation and outcomes’, Frontiers in Medicine, 8. doi:10.3389/fmed.2021.711809.

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K4719013 short</partinfo>
 ==Sequence and Features==
 <partinfo>BBa_K4719013 SequenceAndFeatures</partinfo>
 ==Introduction==
-Vilnius Lithuania iGEM 2023 team's goal was to create a universal synthetic biology system in ''Komagataeibacter xylinus'' for ''in vivo'' bacterial cellulose polymer composition modification. Firstly, we chose to produce a cellulose-chitin polymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by ''K. xylinus''.
+<b>Vilnius-Lithuania iGEM 2023</b> team's goal was to create <b> synthetic biology tools for <i>in vivo</i> alterations of <i>Komagataeibacter xylinus</i> bacterial cellulose polymer composition</b>. Firstly, we chose to produce a <b>cellulose-chitin copolymer</b> that would later be deacetylated, creating <b>bacterial cellulose-chitosan</b>. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed <b>indigo-dyed cellulose</b> that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a <b>bacterial cellulose and polyhydroxybutyrate (PHB) composite</b>, which is synthesized by <i>K. xylinus</i>.
 <br>
 <br>
-Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. We employed two strategies to produce this material. The first approach was to add N-acetylglucosamine into the growth medium [https://parts.igem.org/Part:BBa_K4719013 BBa_K4719013], and the second one was the production of N-acetylglucosamine by ''K. xylinus'' from simple sugars such as glucose, fructose, and saccharose in the growth medium [https://parts.igem.org/Part:BBa_K4719014 BBa_K4719014].
+<b>This specific composite part was used for bacterial cellulose-chitin polymer production.</b> It was achieved by <b>increasing the production of UDP-N-acetylglucosamine</b>, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer.
+We employed two strategies to produce this material: <br>
+<b>1.</b> The first approach was to add N-acetylglucosamine into the growth medium [https://parts.igem.org/Part:BBa_K4719013 BBa_K4719013].<br>
+<b>2.</b> Second one was the production of N-acetylglucosamine by <i>K. xylinus</i> from common sugars such as glucose, fructose, and sucrose in the growth medium [https://parts.igem.org/Part:BBa_K4719014 BBa_K4719014].
 ==Usage and Biology==
-This construct produces an excess of UDP-N-Acetylglucosamine (GlcNAc) in ''K. xylinus'', which can be used by cellulose synthase to produce bacterial cellulose-chitin polymer. Incorporation of N-acetylglucosamine has been previously demonstrated by M. H. Tan (2019) and V. Yadav (2010). ''K. xylinus'' requires genetic modification in order to incorporate GlCNAc into the bacterial cellulose as the natural conversion to UDP-GlcNAc is not efficient. The genes in this composite code for proteins that are responsible for the uptake of extracellular GlcNAc and conversion to UDP-GlcNAc. Therefore, the growth medium has to be supplemented with GlcNAc.
+This construct produces an excess of UDP-N-acetylglucosamine (GlcNAc) in ''K. xylinus'', which can be used by cellulose synthase to produce bacterial cellulose-chitin polymer. Incorporation of N-acetylglucosamine into cellulose has been previously demonstrated by M. H. Tan (2019) and V. Yadav (2010). ''K. xylinus'' requires genetic modification to incorporate GlcNAc into the bacterial cellulose as the natural conversion to UDP-GlcNAc is inefficient. The genes in this composite code for proteins that are responsible for the uptake of extracellular GlcNAc and conversion to UDP-GlcNAc. Therefore, the growth medium has to be supplemented with GlcNAc.
+<br>
-Bacterial cellulose-chitin copolymer has applications in the biomedicine field due to ''in vivo'' biodegradability by the lysosome. However, this copolymer is an intermediate step in your biological system since we chose to improve the qualities of the material by deacetylation. This second enzymatic step produces a bacterial cellulose-chitosan copolymer that has the added benefits of antibacterial activity and amino groups for easier functionalization.
+Bacterial cellulose-chitin copolymer has applications in the biomedicine field due to ''in vivo'' biodegradability by the lysozyme. However, this copolymer is an intermediate substance in our system since we chose to improve the qualities of the material by deacetylation. This second enzymatic step produces a cellulose-chitosan copolymer with the added benefits of antibacterial activity and primary amino groups for easier functionalization.
+<br>
-Since polymer production occurs in ''K. xylinus'' it requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use [https://parts.igem.org/Part:BBa_K1321313 BBa_K1321313] as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool for ''Komagateibacter'' species. We performed PCR of the plasmid eliminating mRFP in order to preserve Anderson promoter J23104 [https://parts.igem.org/Part:BBa_J23104 BBa_J23104], RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] and terminator [https://parts.igem.org/Part:BBa_B0015 BBa_B0015]. ''AGM1'', ''NAG5'' and ''UAP1'' were assembled into the backbone by Gibson assembly.
+<html>
+Since polymer production occurs in <i>K. xylinus</i>, it requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use  <a href="https://parts.igem.org/Part:BBa_K1321313">BBa_K1321313</a> as it was characterized by iGEM14_Imperial team as the most suitable plasmid backbone for <i>Komagateibacter</i> species. We performed PCR of the plasmid eliminating mRFP in order to preserve Anderson promoter J23104 (<a href="https://parts.igem.org/Part:BBa_J23104">BBa_J23104</a>), RBS (<a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and terminator (<a href="https://parts.igem.org/Part:BBa_B0015"> BBa_B0015</a>). <i>AGM1</i>, <i>NAG5</i> and <i>UAP1</i> genes were assembled into the backbone by Gibson assembly.
+</html>
 ==Experimental characterisation==
 <html>
 <body>
 <h3>Polymer production</h3>
 <p>
-Bacterial cellulose-chitin polymer is synthesized by ''K. xylinus'' grown in the Glucose Yeast Extract broth (GYB) while shaking at 180 rpm at 28&deg;C. Varying concentrations of glucose and N-acetylglucosamine have been added to determine the best combination of growing conditions. We determined the carbon source ratio of 1% glucose and 1% N-acetylglucosamine as optimal.
+<b>Bacterial cellulose-chitin polymer</b> is synthesized by <i>K. xylinus</i> grown in glucose yeast extract broth (GYB) while shaking at 180 rpm at 28&deg;C for 7 days. Varying concentrations of glucose and N-acetylglucosamine have been added to determine the best combination of growth conditions. We experimentally determined that the optimal carbon source ratio is 1 % glucose and 1 % N-acetylglucosamine for the highest incorporation of chitin monomers into bacterial cellulose.
 <html>
 </p>
 <figure>
-<div class = "center">
+<div class = "center" >
-<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/3genu-konstruktui.jpg" style = "width:400px;"></center>
+<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/3genu-konstruktu-pt1.jpg" style = "width:400px;"></center>
 </div>
-<figcaption><center>Figure 1:A - bacterial cellulose control group, grown on 2% glucose. B - bacterial cellulose-chitin copolymer, grown on 1% glucose and 1% N-acetylglucosamine.</center></figcaption>
+<figcaption><center><b>Figure 1. Bacterial cellulose-chitin copolymer. A</b> - bacterial cellulose control group, grown on 2 % glucose. <b>B</b> - bacterial cellulose-chitin copolymer, grown on 1 % glucose and 1 % N-acetylglucosamine. The morphology of copolymer is different from natural bacterial cellulose, with a looser association of pellicles. This is consistent with previous work done by V. Yadav et al. (2010).</center></figcaption>
+</figure>
+<p/>
+<h3>Gene expression analysis by RT-qPCR</h3>
+In order to verify synthetic operons <i>AGM1-NAG5-UAP1</i> expression in <i>K. xylinus</i>, we performed <b>real-time quantitative PCR</b>. We were unable to conduct a quantitative analysis of the results due to a shortage of time for generating biological replicates. But, we could compare cycle threshold (Ct) values. This measurement shows how many PCR cycles are required to obtain positive results (Bonacorsi et al., 2021). Expression of every selected gene was detectable because all Ct values were lower than 37.
+<figure>
+<div class = "center" >
+<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/qpcr-3g.png" style = "width:400px;"></center>
+</div>
+<figcaption><center><b>Figure 2. RT-qPCR Ct values for <i>AGM1-NAG5-UAP1</i>.</b> As expected, Ct value for the housekeeping gene of DNA gyrase subunit B (<i>gyrB</i>) is lower compared to the operon, indicating a higher expression. When comparing expression between genes in <i>AGM1-NAG5-UAP1</i>, we noticed that Ct values are very similar for all three genes, meaning that the whole operon is transcribed. </center></figcaption>
+</figure>
+<h3>FTIR spectra of bacterial cellulose-chitin copolymer</h3>
+<figure>
+<div class = "center" >
+<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/3g.png" style = "width:300px;"></center>
+</div>
+<figcaption><center><b>Figure 3. FTIR spectra of bacterial cellulose-chitin copolymer. A</b> - natural bacterial cellulose grown on 2 % glucose (control group). <b>B</b> - bacterial cellulose-chitin copolymer produced by unmodified <i>K. xylinus</i> grown on 1 % glucose and 1 % N-acetylglucosamine. <b>C</b> -  modified <i> K. xylinus</i> producing bacterial cellulose-chitin composite grown on 1 % glucose and 1 % N-acetylglucosamine. </center></figcaption>
 </figure>
 <p>
+The adsorption band at 1589 cm<sup>-1</sup> closely corresponds with OH deformation vibration as described in the literature [3]. This vibration could be attributed to 2’ OH and is the most apparent in the A curve of bacterial cellulose polymer control group. The absorption intensity of this peak decreases with the incorporation of N-acetylgluscosamine. The second band at 1651 cm<sup>-1</sup> is amine I vibration, confirming the incorporation of N-acetylglucosamine [4]. However, the wavenumbers of bands of OH deformation vibration and amine I vibration fall into the same region, therefore, the 1651 cm<sup>-1</sup> band in the control group of bacterial cellulose is visible. With this in mind, we can compare the composition of polymers by observing how both bands change respectively.<br>
+Since FTIR is a qualitative measure, <b>both B and C curves show that the polymer composition has changed from natural bacterial cellulose to bacterial cellulose-chitin copolymer.</b>
+</p>
+<h3>Fluorescence analysis of N-acetylglucosamine incorporation</h3>
+<p>
+To determine if the polymer contains N-acetylglucosamine residues, we used fluorescent lectin staining with wheat germ agglutinin (WGA), Alexa Fluor™ 680 conjugate selectively binding to N-acetylglucosamine. Therefore, natural cellulose should not give a visible signal at this wavelength.<br>
+Construct for incorporation of N-acetylglucosamine into bacterial cellulose <b>improved fluorescence signal significantly</b>, showing consistent distribution of the target monomers.
+<html>
+</p>
+<figure>
+<div class = "center" >
+<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/3g-floresencija.jpg" style = "width:600px;"></center>
+</div>
+<figcaption><center><b>Figure 4. Fluorescent lectin staining with WGA. A</b> - bacterial cellulose grown on 2 % glucose (control group). <b>B</b> - bacterial cellulose control group grown on 1 % glucose and 1 % N-acetylglucosamine. <b>C</b> - bacterial cellulose-chitin copolymer grown on 1 % glucose and 1 % N-acetylglucosamine. Fluorescence signal intensity corresponds with the amount of N-acetylglucosamine in the polymer, therefore unmodified <i> K. xylinus </i> is capable of slight incorporation of N-acetylglucosamine. However, <b>our composite part improves this process significantly</b>. </center></figcaption>
+</figure>
+</p>
+<h3>Chracterization of polymer surface with SEM</h3>
+<p>
+For further verification of bacterial cellulose-chitin polymer structural differences from natural bacterial cellulose, we performed <b>scanning electron microscopy (SEM)</b>.
+<html>
+</p>
+<figure>
+<div class = "center" >
+<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/screenshot-2023-09-21-191920.png" style = "width:600px;"></center>
+</div>
+<figcaption><center><b>Figure 5. Bacterial cellulose-chitin copolymer surface characterization by SEM. A</b> - bacterial cellulose grown on 2 % glucose (control group). <b>B</b> - bacterial cellulose-chitin copolymer grown on 1 % glucose and 1 % N-acetylglucosamine. The arrangement of fibers is much finer in the copolymer due to altered hydrogen bonding properties between glucose monomers. The crystallinity decreases as the fibers of the polymer do not have such strong intramolecular bonds. </center></figcaption>
+</figure>
+</p>
+<h3>Growth burden</h3>
+<p>
+In order to work with <i>E. coli</i> for designing and testing constructs, the growth burden of said synthetic biology tools has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified <i>E. coli</i> DH5&alpha;. The composite <i>AGM1,NAG5,UAP1</i> did not inhibit the growth of <i>E. coli</i> as seen in Figure 6.
+<figure>
+<div class = "center" >
+<center><img src = "https://static.igem.wiki/teams/4719/wiki/partai/3g-growth-burden.png" style = "width:600px;"></center>
+</div>
+<figcaption><center><b>Figure 6.</b> <i> E. coli</i> growth burden of <i>AGM1,NAG5,UAP1</i> composite. </center></figcaption>
+</figure>
+</p>
 <!-- Uncomment this to enable Functional Parameter display
@@ Line 43: / Line 107: @@
 <partinfo>BBa_K4719013 parameters</partinfo>
 <!-- -->
-===References===
+<body>
-.Teh, M.Y. et al. (2019) ‘An expanded synthetic biology toolkit for gene expression control in acetobacteraceae’, ACS Synthetic Biology, 8(4), pp. 708–723. doi:10.1021/acssynbio.8b00168.
+<h2>References</h2>
+. Teh, M.Y. et al. (2019) ‘An expanded synthetic biology toolkit for gene expression control in acetobacteraceae’, ACS Synthetic Biology, 8(4), pp. 708–723. doi:10.1021/acssynbio.8b00168.
+<br>
+. Yadav, V. et al. (2010) ‘Novel in vivo degradable cellulose-chitin copolymer from metabolically engineered gluconacetobacter xylinus’, Applied and Environmental Microbiology, 76(18), pp. 6257–6265. doi:10.1128/aem.00698-10.
+<br>
+. Socrates, G. Infrared and Raman characteristic group frequencies: tables and charts. John Wiley and Sons, LTD. - 3rd ed., 2002
+<br>
+. Barth, A. (2007) ‘Infrared spectroscopy of proteins’, Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1767(9), pp. 1073–1101. doi:10.1016/j.bbabio.2007.06.004.
 <br>
-.Yadav, V. et al. (2010) ‘Novel in vivo degradable cellulose-chitin copolymer from metabolically engineered gluconacetobacter xylinus’, Applied and Environmental Microbiology, 76(18), pp. 6257–6265. doi:10.1128/aem.00698-10.
+. Bonacorsi, S. et al. (2021) ‘Systematic review on the correlation of quantitative PCR cycle threshold values of gastrointestinal pathogens with patient clinical presentation and outcomes’, Frontiers in Medicine, 8. doi:10.3389/fmed.2021.711809.