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• Vanillin synthesis: DNA not yet complete/in library [http://partsregistry.org/wiki/index.php/Part:BBa_I742140]
  • Combination of 5 genes; 3 are available
    • Edinburgh Vanilla production (Pathway synthesis, Edinburgh iGEM 2007)
  • [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17437627#id538997 Vanillin production using metabolically engineered Escherichia coli under non-growing conditions], Ferulic accid --> Vanillin, 5k bp

• Biobricks for vanillin synthesis
  • Send mail to University of Tuscia [Done]
  • Order:
    • (sam8 (tyrosine-ammonia lyase) coding sequence) [ordered]
    • (sam5 (coumarate hydroxylase) coding sequence) [ordered]
    • (COMT gene with ribosome binding site) [Available in kit]

Extra info on vanillin synthesis

• http://www.ncbi.nlm.nih.gov/pubmed/16232583 1 B C Okeke and V Venturi ,Construction of recombinants Pseudomonas putida BO14 and Escherichia coli QEFCA8 for ferulic acid biotransformation to vanillin, J Biosci Bioeng. 1999; 88(1): 103–106.
  
• http://www.ncbi.nlm.nih.gov/pubmed/14602615 2 Jörg Overhage, Alexander Steinbüchel, and Horst Priefert,Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli, Appl Environ Microbiol. 2003 November; 69(11): 6569–6576. doi: 10.1128/AEM.69.11.6569-6576.2003.
  
• http://www.ncbi.nlm.nih.gov/pubmed/10616715 3 J Overhage, H Priefert, J Rabenhorst, and A Steinbüchel,Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene, Appl Microbiol Biotechnol. 1999 November; 52(6): 820–828.
  
• http://www.labmeeting.com/paper/2776259/yoon-enhanced-vanillin-production-from-recombinant-e-coli-using-ntg-mutagenesis-and-adsorbent-resin 4 Yoon SH, Lee EG, Das A, Lee SH, Li C, Ryu HK, Choi MS, Seo WT, and Kim SW, Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin, Biotechnology progress 23(5):1143.
  
• http://www.scielo.br/scielo.php?pid=S1517-83822003000500037&script=sci_arttext 5 Attilio ConvertiI; Danilo de FaveriI; Patrizia PeregoI; Paolo BarghiniII; Maurizio RuzziII; Luciane SeneIII, Vanillin production by recombinant strains of Escherichia coli, Braz. J. Microbiol. vol.34 suppl.1 São Paulo Nov. 2003
  
• http://www.springerlink.com/content/bj47xnqn5kr65wvp/fulltext.pdf 6 S. Achterholt, H. Priefert, and A. Steinbüchel, Identification of Amycolatopsis sp. strain HR167 genes, involved in the bioconversion of ferulic acid to vanillin, Appl Microbiol Biotechnol. 2000 December; 54(6): 799–807
  

• Focus on the last two parts in the synthesis of vanillin since that pathway has been proven
  • Can always add additional 3 paths

• Send mail to U. Edinburgh for the proteins for vanillin synthesis [Done]
  • Received mail back from Edinburgh, biobricking of the parts should still be done. Will receive answer next week

• sam5; no part number, Pst1 site removed
• sam8; coding sequence without RBS
• COMT; coding sequence without RBS
• ech; RBS + ech, in ,
• fcs; RBS + fcs, in ,

• Modeling:
  • Searching for reaction kinetics parameters: degradation of enzymes.

• RBS + ech, in ,
• RBS + fcs, in ,

They were put in the freezer and will be incorporated into competent cells, so they can be multiplied and stored.

• RBS + ech, in ,
• RBS + fcs, in ,

The cells were then transferred to liquid LBmedium to recuperate from the procedure. After a few hours they were transferred to LB/amp plates and grown overnight at 37°C.

• sam5 + RBS in (with PstI site removed),
• sam8 + RBS in ,
• sam8 in coding sequence,
• COMT in coding sequence,

The cells were put into liquid LB to recuperate from the procedure, transferred onto LB/amp plates and grown overnight at 37°C.

We also made liquid cultures of the RBS + fcs and RBS + ech cultures so we can miniprep them tomorrow.

Plasmid DNA from the ech and fcs liquid cultures was isolated today. It yielded respectively 108,8 and 113,9 ng/µl.

• RBS + sam5 conc: 101,3 ng/ul
• RBS + sam8 conc: 95.7 ng/ul
• sam8 conc: 81,8 ng/ul
• COMT , conc: 122,9 ng/ul

Restriction digestion
A1: sam8, EcoRI en SpeI
A2: sam5, EcoRI en XbaI

B1: RBS, EcoRI en SpeI
B2: COMT, EcoRI en XbaI

C1: fcs, EcoRI en SpeI
C2: ech, EcoRI en XbaI

The mixture was incubated in 37°C for 1h30...

...followed by gel electrophoresis of the digested parts

A1 and A2 showed a very low concentration and the 260/280 value was quite high so both were electrophoresed again. B1 (RBS) had failed because it was too short, however ligating B1 and B2 (COMT) was no longer needed since the part with RBS was available in the iGEM 2009 kit .

A1 and A2 were redigested:
A1: sam8, EcoRI en SpeI
A2: sam5, EcoRI en XbaI
followed by gel electrophoresis

• COMT was grown
• sam5 and sam8 were ligated
• ech and fcs were ligated

• Ligated genes are electrophoresed.
• Reorderd part (COMT + RBS) from the registry.
• Also we realized the proteins need to be degraded fast for the control mechanism to work, so a ssRA degradation tag like ANDENYALVA or ANDENYALAA is needed.
  • However since some genes have just been ligated we will only tag sam5, COMT and the ech gene.

• There was no growth of sam8/sam5, fcs/ech and COMT plates.
  • Possible reason is a problem with the electrocompetent cells
  • Or the concentration of sam5/sam8 is too low
• Plan for tomorrow is to create new electrocompetent cells
  • Prepared LB and glycerol today
• Made liquid cultures sam8, sam5, fcs and ech
• Made new agarplates with Ampicilin

• Miniprep on sam5, sam8 and fcs
  • There was no growth on ech, thus new cultures in liquid medium
• Restriction digest on sam5, sam8 and fcs
• Gel electrophoresis on sam5, sam8 and fcs
• Gel extraction on sam5 and fcs

Nanodrop results:

Notes:

• sam8 showed a very low concentration on the gel electrophoresis. Thus, it is better to redo this test.
• 260/280 for fcs was very high and the concentration was not high enough. Therefore, we will redo the extraction and use multiple tubes on the same filter to increase the concentration

1. Plated COMT (+RBS) from the registry
   • Growing overnight in 37°C
2. Plated ech
   • From newly made liquid culture
3. Miniprep of ech and sam8
   • Nanodrop results:

1. Restriction digest on ech and sam8
2. Gel electrophoresis on ech and sam8
3. New electrocompetent cells were used for the electroporation with the old ligations
   • Lig A -> sam5 + sam8
   • Lig C -> ech + fcs
   • Note: there was no spark
4. Plating of the electroporated cells
   • 200 μl per plate twice
   • 600 μl left of A and C

1. Gel extraction of the 4 lanes from sam8 () and ech ()
   • To test an additional blanc, we extracted a blanc piece of gel
   • Nanodrop results were done with the conventional blanc (just elution buffer) and with the extracted blanco gel. However, since the results between both varied too much and the extracted blanco gel was not significantly better, we decided to use the conventional blanco in order to compare between former nanodrop measurements

Nanodrop:

• Ligation of sam5 (from Wednesday) + sam8 (extracted today)

• Ligation of ech and fcs. For fcs we took the digest from last week (C1), because this week's restriction digest had a very low concentration (4,2) and a very high 260/280 (6,08)

• Cells were stored at 16°C

• Plated the electroporated cells from the A and C ligation
• Made Agar and poured new plates with Ap

• SAMS (ligation sam5+sam8) and EF (ligation ech+fcs): electroporated ...
• and plated out

• Cells didn't grow. Yet again...
• Re-plated the ligation products (SAMS and EF)

• We propose 3 tests to check if the restriction digest (esp. between EcoRI and XbaI, because they lie very close together) works properly:

Test 1: Cut every plasmid (sam8, sam5, ech, Fcs) separately with EcoRI, XbaI and -only sam8 and fcs- with SpeI.

Test 2: Cut subsequently with EcoRI and XbaI, leaving an incubation period of 1h between two digests. Total volume: 30µl, to dilute glycerol (which inhibits restriction)

Test 3: Use pUC18 vector

• Made fluid cultures of sam8, sam5, ech, fcs

• Miniprep sam8, sam5, ech, fcs plasmids in triple
• Nanodrop concentrations:

• We took the plasmids with the lowest concentrations to perform the restriction test, described above. Afterwards, we ran the restriction products, together with uncut plasmids, on a gel.
• Calculations for restriction:

• Meanwhile ... one plated EF colony seemed to have grown. Made fluid culture of EF.

• EF colonies present! So, we took 1ml off for further growth and miniprepped the remaining 4ml in triple
• Nanodrop concentrations:

• We performed a digest with EcoRI and SpeI to cut out the insert and check it's length. Enzymes were added separately, with 1h in between. Total volume was 30µl.
• Calculations restriction

• In the afternoon, the remaining 1ml was re-plated and put on 37°C

2)

• Test 1: Results from yesterday's restriction test were inconclusive, because the DNA ladder didn't run properly so the length of the fragments couldn't be read. Therefore, we did it again...
• Restriction=OK! Test 1 completed.

3)

• Test 2: cutting with 2 enzymes subsequently (1h incubation in between) in a volume of 30µl. Using a larger volume should dilute the glycerol, which inhibits cutting. After that, the products are incubated overnight to allow full restriction. sam8 and fcs are cut with EcoRI and SpeI; sam5 and ech are cut with EcoRI and XbaI

• Calculations restriction

• Test 2: We loaded yesterday's restrictions on an agarose gel. It showed that the enzymes cut properly.

However, the EF results were unexpected. It seems that somehow the bacteria had taken in a self-closed ech-plasmid. The signals do not correspond with an EF-ligation product.

• The DNA was purified from the gel

Nanodrop results:

• sam8 and sam5; fcs and ech were ligated

Used volumes: