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• Vanillin synthesis: DNA not yet complete/in library [1]
  • Combination of 5 genes; 3 are available
    • Edinburgh Vanilla production (Pathway synthesis, Edinburgh iGEM 2007)
  • 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

• [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.
  
• [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.
  
• [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.
  
• [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.
  
• [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
  
• [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:

• Electroporation competent cells with EF and SAMS
• Cells were plated out and grown overnight

• We have colonies!!
• Lots of EF, enough of SAMS
• We made fluid cultures and let them grow overnight

• Miniprep of fluid cultures

Nanodrop results:

• Restriction:

-We performed 2 simultaneous digests: one to verify the length of the product (Restriction2Test) using SAMS II and EF III and another to prepare for ligation (Restriction4Real) with promotor and terminator using SAMS III and EF II

-We cut with the 2 enzymes separately, allowing a one hour incubation in between. EF: E/S SAMS: E/S

-Restriction volumes:

Restriction2Test

Restriction4Real

• Terminator was also digested

Terminator: E/X

• Restriction2Test: Agarose gel electrophoresis to validate if the test samples have the correct length. Signals appeared that could not be explained

• Restriction4Real: we performed a gel electrophoresis together with opened plasmids (cut with EcoRI) to compare sizes

• Gel signals were inconclusive: gel ran not long enough

• We had to redo yesterday's test with the same volumes, allowing the gel to run longer
  • EF ligation was too short
  • Restriction digest for new ech and fcs parts (and also sam5 and sam8)
  • Put overnight in 16°C

• Split the restriction digest in 2 parts
  • 1 Part was directly used for ligation using double the mass required(SAMS II and EF II)

• The 2nd part was first put on a gel electrophoresis and purified before it was used for ligation
• Gel electrophoresis of restriction on sam5, sam8, fcs and ech
  • sam5 and sam8 showed expected results
  • fcs and ech had unexpected signals
    • fcs showed only 1 result on gel, expected two
    • ech showed 4 results in gel (all too short), expected one
  • sam5 and sam8 were purified and ligated (SAMS I)

• ech and fcs were cut again and put overnight (over the weekend...) in 16°C

• Electroporation of SAMS I, SAMS II AND EF II
• Restriction digest of ech and fcs from Saturday was tested and showed 6 signals with varying lengths for ech and 1 very large signal for fcs, both inconsistent. To test for errors in the restriction sites we cut each vector with 1 restriction digest for each of the sites. Giving a total of 4 tests for ech and 4 for fcs.
  • Restriction at 37°C overnight.
• For ligation of SAMS III RD (from Thursday) and TER II we need 44,4 µl of SAMS III which we don't have thus we cut two additional volumes with SAMS I overnight
  • SAMS I A RD and SAMS I B RD

• SAMS I B RD is used for ligation with TER II RD
  • Since we want to avoid gel purification, we use the direct sample from the restriction digest
  • The restriction enzymes are denatured by keeping the sample at 85°C for 20 minutes

• The genes fcs and ech are individually cut with the four restriction enzymes

• Results on gel show that all the genes have the same length and display 1 line.
• SAMS I A RD is put on gel, to be used for gel extraction and a part of SAMS I B RD is put on gel to check the length.
  • No signal... grrr...
• Replated colonies from the original SAMS plate from 24/8
  • Took 4 individual colonies and plated them on 4 corners of a petri dish

1. the 4 genes(sam5, sam8, ech, fcs) where taken from the original DNA that was sent, electroporated and plated. This in order to make a glycerol stock.

• We also started another method to try and ligate the different genes for vanillin production by using PCR. Today we did PCR on the ech, fcs, sam5 and sam8 genes to get the DNA out of the plasmid. We used the prefix and suffix primers from last year, these primers keep the pre- and suffix of the biobrick part intact and allow us to work with the purified biobricks themself.

• The four grown colonies are put on liquid culture
• New (again...) restriction digest on sam5, sam8, fcs and ech, genes were miniprepped on Tuesday.
  • Overnight at 16°C
• The plates with sam5, sam8, ech and fcs were re-ented. From sam5 and fcs four colonies were picked and plated.

• The PCR from yesterday did not deliver any results, probably because of an error when preparing the PCR mix. A new PCR was prepared and started today.

NOT PCR

• Electroporation of SAMS+TER ligation (from Tuesday ligation... the one that looked bad....)
• Miniprepped the 4 colonies from SAMS

• Restriction digest of SAMS
• Gel electrophoresis of restriction from ech, fcs, sam5 and sam8
  • Result looks great (woohoo!)
  • Cut and purified from gel

• Used ech and fcs to ligate using 5 μl from ech and 23,5 μl from fcs. There was not enough sam8 for ligation. Redo restriction at some point in the future...
• 4 colonies from sam8 and ech were picked and plated

PCR

• The PCR from yesterday went very well, we had a huge amount of amplified biobrick DNA of the different parts. Agarose gel electrophoresis was used to test if the different pieces of DNA created by PCR had the correct length.

• Amplified DNA was purified and cut with different restriction enzymes. After restriction, DNA was purified again before ligation.

• After the restriction, sam5, sam8 and terminator were ligated in a three-way ligation. The same was done for ech, fcs and terminator. We also started a ligation of just sam5 and sam8 and ech and fcs.

• The restriction digest from SAMS and the terminator was put on gel
  • It appears there is no SAMS in between the vector, the length is approx. 1500 bp which can only be sam5 or sam8
• Electroporation of the ligation EF
  • Plated and put at 37°C
• Liquid cultures made from sam5, sam8, ech and fcs.

• Restriction digest overnight of sam5 (1,2) and sam8 (1,2)
• Growth of EF Ligation (woohoo!)
  • Transferred 4 colonies from EF ligation to a new plate
  • Put the 4 transferred colonies on liquid medium

A miniprep and restriction digest (ON) of sam5, sam8, ech and fcs was performed on Saturday. On Sunday, a gel was run to check the inserts. These were good so a motherplate for all the genes could be made.

• Miniprep of the four colonies from EF ligation
  • Accidentally used double volumes of elution buffer on EF4

• Gel electrophoresis from restriction on sam5 and sam8 looked good
• Purified and ligated the restriction from sam5 and sam8 overnight
  • Concentration of sam8 was barely enough for ligation...

• Restriction digest on EF1, EF2, EF3 and EF4
  • Gel electrophoresis of digestion, result: EF1 and EF3 appear to have an insert. The insert of EF4 is too short, between 1500 and 2000 bp (might just be ech) and EF2 appears to have been cut by only 1 enzyme
  • Also, restriction digest overnight results in an extra very weak intensity band at 600 and one between 1500 and 2000 bp
    • This result has been seen before on just ech. It might be due to an extra restriction site on the ech gene. The two bands could result from this same band. The addition of both bands would then be the same as the insert.
• Electroporation of SAMS1 and SAMS2
• Glycerolstock for sam5, sam8, ech and fcs

• Restriction digest on Terminator
• Growth on SAMS plates (woohoo!)
• Transferred 4 colonies from the SAMS plates (SAMS1, SAMS1 pellet, SAMS2, SAMS2 pellet) to a new plate
• Gel electrophoresis of all the 8 EF restriction digests (2 per colony)
  • Cut and purified EF1 and EF3

• Gel electrophoresis of TER restriction
  • The vector for the TER is Psb1AK3 and has 3100bp, with an insert of ±3200 bp
  • Gel extraction

• Created a motherplate from EF1
• Ligated EF restriction + TER restriction
• Transferred the SAMS 1 and SAMS 2 colonies to liquid medium

• Miniprep SAMS

• Restriction SAMS + Promotor (
  • Promotor restriction results in 2 parts of 800 bp and 2k bp
• EFT electroporation no result, try again on monday...