Team:Saarland/Test

From 2014.igem.org

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<h1> 3. Find allies and join forces </h1>
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<h1> Labbook </h1>
<br>
<br>
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<h2><i>B. megaterium </i> </h2>  
+
<h2> February</h2>
 +
*Develop strategy for the first ever biotechnological production of the naked mole rat´s high molecular mass hyaluronic acid and the characterisation of its inhibitory effects on carcinogenesis.
 +
*Recruiting committed team members.
 +
 
 +
<h2> March </h2>
 +
*Looking for sponsors.
 +
*Team registration.
 +
 
 +
<h2>April</h2>
 +
*Looking for sponsors
 +
*Design of a team logo
 +
 
 +
<h2>May </h2>
 +
*Looking for sponsors
 +
*Conception and design of the team wiki
 +
 
 +
*Waiting for completion of <i>has</i>2 gene synthesis.
 +
 
 +
<h3>Highlight at Week 4</h3>
 +
*Meet up of German iGEM teams in Munich. Inspiring Weekend. We enjoyed the short trip.
 +
 
 +
<h2>June</h2>
 +
<h3>1. Week</h3>
 +
*Finally, the synthesised <i>has</i>2 gene has arrived!
 +
 
 +
*Transformation of electro competent <i>E. coli</i> cells with Eurofins standard delivery plasmid pexK4 containing the synthesised <i>has</i>2 insert DNA.
 +
 
 +
*Plasmid preparation and control digestion to confirm the presence of <i>has</i>2 insert DNA.
 +
 
 +
 
 +
 
 +
<h3>2. Week</h3>
 +
*First attempt to insert <i>has</i>2 DNA into the multiple cloning site (MCS) of pSMF2.1 plasmid construct. This shuttle plasmid allows on the one hand the amplification of plasmid DNA in high copy numbers in <i>E. coli</i> cells. On the other hand pSMF2.1 is optimised for expression of recombinant proteins in our favourite chassis <i>B. megaterium. Has</i>2 insert DNA was absent in transformants.
 +
 
 +
<h3>3. Week</h3>
 +
*Repetition of cloning experiments (week 2) with new T4 ligase provided many <i>E. coli </i>transformants with pSMF2.1 plasmid construct containing <i>has</i>2 insert DNA.
 +
 
 +
<h3>4. Week</h3>
 +
*Extraction of genomic DNA from <i>B. megaterium</i> was performed.
 +
 
 +
*Optimal PCR conditions for the amplification of genomic DNA from <i>B. megaterium </i>were tested. First gene (<i>UDP-glc</i>DH) involved in synthesis of precursor molecules and probably contributing to a high yield production of high molecular mass hyaluronic acid was successfully amplified.
 +
 
 +
*First attempt for downstream insertion of <i>UDP-glc</i>DH DNA into the MCS of pSMF2.1 plasmid construct that already contained the <i>has</i>2 insert DNA was successful.
 +
 
 +
<h2>July</h2>
 +
<h3>1. Week</h3>
 +
*Open day at Saarland University. The iGEM team was proud to present its project to the general public.
 +
 
 +
*Plasmid preparation and control digestion to confirm the simultaneous presence of <i>has</i>2 and <i>UDP-glc</i>DH insert DNA in the pSMF2.1 plasmid construct.
 +
 
 +
*Preparation of fresh protoplasts for transformation of <i>B. megaterium</i> strain MS941 was successful. Competence is outstanding.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3>2. Week</h3>
 +
*Transformations of <i>B. megaterium</i> protoplasts with different pSMF2.1 plasmid constructs were successful. Screening process for the identification of <i>B. megaterium</i> transformants with correct plasmid constructs was time consuming.
 +
 
 +
*<i>B. megaterium</i> chassis for the first ever biotechnological production of high molecular mass hyaluronic acid is ready!
 +
 
 +
<h3>3. Week</h3>
 +
*Cultivation of <i>B. megaterium</i> and induction of recombinant protein production were performed. Samples were prepared after an appropriate cultivation time to check and characterise recombinant protein expression levels of Has2 and UDP-GlcDH.
 +
 
 +
*These samples were also used for viscosity measurement since the outstanding water binding capacity of high molecular mass hyaluronic acid should increase the viscosity of the cultivation medium.
 +
 
 +
<h3>4. Week</h3>
 +
*Illegal restriction sites within <i>has</i>2 and <i>UDP-glc</i>DH were changed via QC-PCR. Resulting biobrick DNAs were inserted into the standard biobrick plasmid construct pSB1C3.
 +
 
 +
*Measurement of viscosity was performed. There was no difference in viscosity of the culture medium and consequently no high molecular mass hyaluronic acid production detectable in the first attempt.
 +
 
 +
<h2>August</h2>
 +
<h3>1. Week</h3>
 +
*SDS-PAGE
 +
 
 +
*Power failure at whole Saarland University during weekend. Reorder of heat sensitive enzymes including polymerases, restriction enzymes and ligase.
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
 +
 
 +
<h3>2. Week</h3>
 +
*Functional enzymes arrived.
 +
 
 +
*Repetition of all <i>B. megaterium</i> transformations during timeout since corresponding glycerol stocks were damaged.
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
 +
 
 +
 
 +
<h3>3. Week</h3>
 +
*Generation of <i>has2-gfp</i> fusion gene via SOE-PCR for quick evaluation of Has2 expression level and localisation in <i>B. megaterium</i> was performed. Insertion of <i>has2-gfp</i> fusion gene into MCS of pSMF2.1 plasmid construct was successful. A lot of positive <i>E. coli</i> transformants!
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
 +
 
 +
 
 +
<h3>4. Week</h3>
 +
*Plasmid preparation and transformation of fresh prepared <i>B. megaterium</i> protoplasts with pSMF2.1 plasmid construct containing the <i>has2-gfp</i> fusion gene was successful.
 +
 
 +
*Expression of Has-GFP fusion protein and samples for localisation studies via fluorescence microscopy were prepared.
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
 +
 
 +
 
 +
<h2>September</h2>
 +
<h3>1. Week</h3>
 +
*Meet and greet with our sponsors. Thank you very much for your great support!
 +
 
 +
*First attempt to confirm Has-GFP expression via epi fluorescence microscopy was successful. <i>B. megaterium</i> cells glew beautifully green.
 +
 
 +
*Additional genes involved in synthesis of precursor molecules were successfully amplified from genomic DNA of <i>B. megaterium</i> and inserted into the standard biobrick plasmid construct pSB1C3.
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
 +
 
 +
* Evaluation of results and creation of figures for our team wiki.
 +
 
 +
 
 +
<h3>2. Week</h3>
 +
*Expression of Has-GFP fusion protein was repeated and samples for localisation studies via laser scanning microscopy were prepared. No fluorescence signal was detectable after cultivation under the same conditions.
 +
 
 +
*Illegal restriction sites within the genes for pathway engineering were changed via QC-PCR.
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
 +
 
 +
*Evaluation of results and creation of figures for our team wiki.
 +
 
 +
 
 +
 
 +
<h3>3. Week</h3>
 +
*Expression of Has-GFP fusion protein was tested under different cultivation conditions. No significant fluorescence signal. Possible misfolding of Has-GFP protein
 +
 
 +
*Sequencing of biobricks was prepared.
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
 +
 
 +
*Evaluation of results and creation of figures for our team wiki.
 +
 
 +
 
 +
<h3>4. Week</h3>
 +
*Waiting for sequencing results.
 +
 
 +
*Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
 +
 
 +
*Evaluation of results and creation of figures for our team wiki.
 +
 
 +
 
 +
<h2>October</h2>
 +
<h3>1. Week</h3>
 +
*Biobrick sequencing was finished. Biobricks are ready to ship to the iGEM Headquarter.
 +
 
 +
*Human Practices: Beta test of the roleplaying game for the ethical and social implications of our project.
 +
 
 +
*Evaluation of results and creation of figures for our team wiki.
 +
 
 +
<h3>2. Week</h3>
 +
*Last modifications on team wiki!
 +
 
 +
*Human Practices: Last modifications of the roleplaying game for the ethical and social implications of our project.
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<!--Bild B.megaterium -->
 
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<br>
 
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<i>B. megaterium </i> belongs to the group of Gram-positive, aerobic bacteria. It’s typically found in soil, but can also survive in sea water or dried foods (Vary <i>et al</i>., 2007). Since its discovery in 1884, it has become one of the most important organisms for industrial and research purposes, because it combines numerous useful characteristics. Due to its extraordinary size of up to 1,5 µm x 4 µm and it`s impressive cell volume of more than 60 µm3, <i>B. megaterium</i>is predestined for studies regarding cell structure and protein localisation (Boyke <i>et al</i>., 2010; Vary <i>et al</i>., 1992). Furthermore it is able to metabolise more than 62 carbon sources, making its cultivation efficient and inexpensive (Millet <i>et al</i>., 1962). Probably the most useful characteristic for our purposes is its ability of preserving plasmids over multiple generations, making it a good specimen for the secretion of proteins and other organic molecules. Especially concerning the <i>B. megaterium</i> strain MS941 that was derived from the strain DSM319 by knockout of the neutral protease gene nprM (Wittchen <i>et a </i>l. 1995). For this reason the non sporulating strain MS941 is ideal for heterologous protein expression. This was shown by overexpression of GFP (Stammen <i> et a </i>l., 2007).<br> <br>
 
-
Additionally, the biosynthetic pathway of hyaluronic acid precursor molecules is already established in <i>B. megaterium</i> since UDP-N-acetyl-D-glucosamine and UDP-D-glucuronic acid are also essential components for cell wall synthesis in Gram-positive bacteria. Manual supplementation of these extremely expensive precursor molecules is not longer necessary. This could contribute to a future profitable biotechnological production of the high molecular mass hyaluronic acid (HMM-HA) of the naked mole rat. Homology searches in the <i>B. megaterium</i> genome have also shown that there are no endogenous hyaluronidases, which would otherwise immediately degrade the produced hyaluronic acid. Furthermore <i>B. megaterium</i> does not possess an endogenous hyaluronan synthase (Has). For this reason our team can be sure that hyaluronic acid has exclusively been synthesised by the correct enzyme and features the correct molecular weight and anti carcinogenic properties.<br> <br>
 
-
For the optimisation of hyaluronic acid production our team intends to overexpress endogenous <i>B. megaterium</i> proteins that are necessary for the production of hyaluronic acid precursor molecules. We think that the flow equilibrium of metabolites will be shifted towards production of HA, rather than to cell wall components. This approach could make a considerable contribution for high yield production of HMM-HA. The identification of corresponding genes in <i>B. megaterium</i> is based on the biosynthetic pathway for the HA production in group A and group C streptococci as well as for HA production in <i>B. subtilis</i> (Widner <i>et al</i>. 2005). The proposed biosynthetic pathway for production of the HA precursor molecules in <i>B. megaterium</i> after <i>in silico</i> gene homology search on MegaBac v9 platform is shown in figure 2.
 
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<br>
 
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<!-- Bild biosentetic pathway -->
 
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<br>
 
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However, when the HA will be successfully produced as we described in figure 2, then it is supposed to be secreted into the medium. Since studies with the similar<i> B. subtilis</i> expressing a streptococci hyaluronan synthase (HasA) have already shown similar results. It’s either possible that there is a specific transporter for oligosaccharides mediating HA secretion into the medium, or that the multi membrane domains of the hyaluronan synthase themselves form a channel for the secretion of the nascent HA chain (Weigel, 2002)
 
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<br>
 
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<!-- Hyperlink Modelling -->
 
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<!-- Hyperlink Teil drei -->
 
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<br>
 

Revision as of 11:45, 9 October 2014



Labbook


February

  • Develop strategy for the first ever biotechnological production of the naked mole rat´s high molecular mass hyaluronic acid and the characterisation of its inhibitory effects on carcinogenesis.
  • Recruiting committed team members.

March

  • Looking for sponsors.
  • Team registration.

April

  • Looking for sponsors
  • Design of a team logo

May

  • Looking for sponsors
  • Conception and design of the team wiki
  • Waiting for completion of has2 gene synthesis.

Highlight at Week 4

  • Meet up of German iGEM teams in Munich. Inspiring Weekend. We enjoyed the short trip.

June

1. Week

  • Finally, the synthesised has2 gene has arrived!
  • Transformation of electro competent E. coli cells with Eurofins standard delivery plasmid pexK4 containing the synthesised has2 insert DNA.
  • Plasmid preparation and control digestion to confirm the presence of has2 insert DNA.


2. Week

  • First attempt to insert has2 DNA into the multiple cloning site (MCS) of pSMF2.1 plasmid construct. This shuttle plasmid allows on the one hand the amplification of plasmid DNA in high copy numbers in E. coli cells. On the other hand pSMF2.1 is optimised for expression of recombinant proteins in our favourite chassis B. megaterium. Has2 insert DNA was absent in transformants.

3. Week

  • Repetition of cloning experiments (week 2) with new T4 ligase provided many E. coli transformants with pSMF2.1 plasmid construct containing has2 insert DNA.

4. Week

  • Extraction of genomic DNA from B. megaterium was performed.
  • Optimal PCR conditions for the amplification of genomic DNA from B. megaterium were tested. First gene (UDP-glcDH) involved in synthesis of precursor molecules and probably contributing to a high yield production of high molecular mass hyaluronic acid was successfully amplified.
  • First attempt for downstream insertion of UDP-glcDH DNA into the MCS of pSMF2.1 plasmid construct that already contained the has2 insert DNA was successful.

July

1. Week

  • Open day at Saarland University. The iGEM team was proud to present its project to the general public.
  • Plasmid preparation and control digestion to confirm the simultaneous presence of has2 and UDP-glcDH insert DNA in the pSMF2.1 plasmid construct.
  • Preparation of fresh protoplasts for transformation of B. megaterium strain MS941 was successful. Competence is outstanding.



2. Week

  • Transformations of B. megaterium protoplasts with different pSMF2.1 plasmid constructs were successful. Screening process for the identification of B. megaterium transformants with correct plasmid constructs was time consuming.
  • B. megaterium chassis for the first ever biotechnological production of high molecular mass hyaluronic acid is ready!

3. Week

  • Cultivation of B. megaterium and induction of recombinant protein production were performed. Samples were prepared after an appropriate cultivation time to check and characterise recombinant protein expression levels of Has2 and UDP-GlcDH.
  • These samples were also used for viscosity measurement since the outstanding water binding capacity of high molecular mass hyaluronic acid should increase the viscosity of the cultivation medium.

4. Week

  • Illegal restriction sites within has2 and UDP-glcDH were changed via QC-PCR. Resulting biobrick DNAs were inserted into the standard biobrick plasmid construct pSB1C3.
  • Measurement of viscosity was performed. There was no difference in viscosity of the culture medium and consequently no high molecular mass hyaluronic acid production detectable in the first attempt.

August

1. Week

  • SDS-PAGE
  • Power failure at whole Saarland University during weekend. Reorder of heat sensitive enzymes including polymerases, restriction enzymes and ligase.
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project

2. Week

  • Functional enzymes arrived.
  • Repetition of all B. megaterium transformations during timeout since corresponding glycerol stocks were damaged.
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project


3. Week

  • Generation of has2-gfp fusion gene via SOE-PCR for quick evaluation of Has2 expression level and localisation in B. megaterium was performed. Insertion of has2-gfp fusion gene into MCS of pSMF2.1 plasmid construct was successful. A lot of positive E. coli transformants!
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project


4. Week

  • Plasmid preparation and transformation of fresh prepared B. megaterium protoplasts with pSMF2.1 plasmid construct containing the has2-gfp fusion gene was successful.
  • Expression of Has-GFP fusion protein and samples for localisation studies via fluorescence microscopy were prepared.
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project


September

1. Week

  • Meet and greet with our sponsors. Thank you very much for your great support!
  • First attempt to confirm Has-GFP expression via epi fluorescence microscopy was successful. B. megaterium cells glew beautifully green.
  • Additional genes involved in synthesis of precursor molecules were successfully amplified from genomic DNA of B. megaterium and inserted into the standard biobrick plasmid construct pSB1C3.
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
  • Evaluation of results and creation of figures for our team wiki.


2. Week

  • Expression of Has-GFP fusion protein was repeated and samples for localisation studies via laser scanning microscopy were prepared. No fluorescence signal was detectable after cultivation under the same conditions.
  • Illegal restriction sites within the genes for pathway engineering were changed via QC-PCR.
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
  • Evaluation of results and creation of figures for our team wiki.


3. Week

  • Expression of Has-GFP fusion protein was tested under different cultivation conditions. No significant fluorescence signal. Possible misfolding of Has-GFP protein
  • Sequencing of biobricks was prepared.
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
  • Evaluation of results and creation of figures for our team wiki.


4. Week

  • Waiting for sequencing results.
  • Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
  • Evaluation of results and creation of figures for our team wiki.


October

1. Week

  • Biobrick sequencing was finished. Biobricks are ready to ship to the iGEM Headquarter.
  • Human Practices: Beta test of the roleplaying game for the ethical and social implications of our project.
  • Evaluation of results and creation of figures for our team wiki.

2. Week

  • Last modifications on team wiki!
  • Human Practices: Last modifications of the roleplaying game for the ethical and social implications of our project.




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