Team:Saarland/Test

From 2014.igem.org

(Difference between revisions)
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<br>
<br>
<h2> February</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.
*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.
*Recruiting committed team members.
 +
<h2> March </h2>
<h2> March </h2>
 +
*Looking for sponsors.
*Looking for sponsors.
*Team registration.
*Team registration.
 +
<h2>April</h2>  
<h2>April</h2>  
 +
*Looking for sponsors
*Looking for sponsors
*Design of a team logo
*Design of a team logo
 +
<h2>May </h2>
<h2>May </h2>
 +
*Looking for sponsors
*Looking for sponsors
*Conception and design of the team wiki
*Conception and design of the team wiki
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<h3>Highlight at Week 4</h3>
<h3>Highlight at Week 4</h3>
*Meet up of German iGEM teams in Munich. Inspiring Weekend. We enjoyed the short trip.
*Meet up of German iGEM teams in Munich. Inspiring Weekend. We enjoyed the short trip.
 +
<h2>June</h2>
<h2>June</h2>
 +
<h3>1. Week</h3>
<h3>1. Week</h3>
 +
*Finally, the synthesised <i>has</i>2 gene has arrived!  
*Finally, the synthesised <i>has</i>2 gene has arrived!  
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*Plasmid preparation and control digestion to confirm the presence of <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>
<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.
*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>
<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.
*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>
<h3>4. Week</h3>
 +
*Extraction of genomic DNA from <i>B. megaterium</i> was performed.  
*Extraction of genomic DNA from <i>B. megaterium</i> was performed.  
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*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.  
*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>
<h2>July</h2>
 +
<h3>1. Week</h3>
<h3>1. Week</h3>
 +
*Open day at Saarland University. The iGEM team was proud to present its project to the general public.  
*Open day at Saarland University. The iGEM team was proud to present its project to the general public.  
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<h3>2. Week</h3>
<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.
*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.
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<h3>3. Week</h3>
<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.  
*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.  
Line 72: Line 89:
<h3>4. Week</h3>
<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.  
*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.  
Line 77: Line 95:
<h2>August</h2>
<h2>August</h2>
 +
<h3>1. Week</h3>
<h3>1. Week</h3>
 +
*SDS-PAGE  
*SDS-PAGE  
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<h3>2. Week</h3>
<h3>2. Week</h3>
 +
*Functional enzymes arrived.  
*Functional enzymes arrived.  
Line 93: Line 114:
<h3>3. Week</h3>
<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!
*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!
Line 99: Line 121:
<h3>4. Week</h3>
<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.
*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.
Line 107: Line 130:
<h2>September</h2>
<h2>September</h2>
 +
<h3>1. Week</h3>
<h3>1. Week</h3>
 +
*Meet and greet with our sponsors. Thank you very much for your great support!
*Meet and greet with our sponsors. Thank you very much for your great support!
Line 120: Line 145:
<h3>2. Week</h3>
<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.
*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.
Line 131: Line 157:
<h3>3. Week</h3>
<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
*Expression of Has-GFP fusion protein was tested under different cultivation conditions. No significant fluorescence signal. Possible misfolding of Has-GFP protein
Line 141: Line 168:
<h3>4. Week</h3>
<h3>4. Week</h3>
 +
*Waiting for sequencing results.
*Waiting for sequencing results.
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<h2>october</h2>
<h2>october</h2>
 +
<h3>1. Week</h3>
<h3>1. Week</h3>
 +
*Biobrick sequencing was finished. Biobricks are ready to ship to the iGEM Headquarter.
*Biobrick sequencing was finished. Biobricks are ready to ship to the iGEM Headquarter.
Line 157: Line 187:
<h3>2. Week</h3>
<h3>2. Week</h3>
 +
*Last modifications on team wiki!
*Last modifications on team wiki!

Revision as of 11:48, 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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