From 2007.igem.org

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Contents 1 Biobricks Revived 2 Lab Diary 2.1 Thu 12th July 2.2 Fri 13th July 2.3 Mon 16th July 2.4 Tue 17th July 2.5 Wed 18th July 2.6 Thu 19th July 2.7 Fri 20th July 2.8 Mon 23rd July 2.9 Tue 24th July 2.10 Wed 25th July 2.11 Thu 26th July 2.12 Fri 27th July 2.13 Monday 30th July 2.14 Tuesday 31st July 2.15 Wednesday 1st August 2.16 Thursday 2nd August

Biobricks Revived

This is a list of the biobricks we have revived from the registry

Brick	Description	Well	Used for
[http://partsregistry.org/Part:BBa_J04430 BBa_J04430]	IPTG induced GFP	Plate 2, 1L	Test biobrick revival process
[http://partsregistry.org/Part:BBa_E0241 BBa_E0241]	PoPS --> GFP	Plate 2, 15L	For use in dif experement 1
[http://partsregistry.org/Part:BBa_J45700 BBa_J45700]	Wintergreen Scent	Plate 3, 7B	Place into lacto bacillus
[http://partsregistry.org/Part:BBa_E0422 BBa_E0422]	fast degrading ECFP	Plate 1, 11G	For use in dif experement 1
[http://partsregistry.org/Part:BBa_I0500 BBa_I0500]	arabinose promoter	Plate 2, 9I	For use in dif experement 3
[http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510]	CI (Lambda) Inverter	Plate 2, 13K	For use in dif experement 3

Lab Diary

Thu 12th July

• IPTG induced GFP revived ([http://partsregistry.org/Part:BBa_J04430 BBa_J04430])

Test biobrick revival process

Fri 13th July

• Alfalfa Seeds Planted (in Agar)

Growing alfalfa plants, to get leaves from which to extract DNA for the caffeic acid-3-o-methyltransferase gene (part of the vanillin synthesis pathway)

• Biobricks [http://partsregistry.org/Part:BBa_E0241 BBa_E0241] and [http://partsregistry.org/Part:BBa_J45700 BBa_J45700] revived, using modified protocal from open wetware site

Mon 16th July

• Begin DNA extraction from Bamboo

Test DNA extraction process from plants

• Prepared mini preps for GFP and mint genes

Tue 17th July

• Alfalfa Transferred to compost

• DNA containing GFP and mint genes extracted from E.Coli

Wed 18th July

• Double digest GFP and mint mini preps, using restriction enzymes SpeI and EcoRI. These were then run on a polyacramide gel
• Biobrick ECFP [http://partsregistry.org/Part:BBa_E0422 BBa_E0422] revived and transformed into E.coli JM109 competent cells

Thu 19th July

• Repeat of double digest (using SpeI & EcoRI) of GFP and mint mini preps with increased concentration of DNA
• Replate colonies of ECFP, 4 overnight cultures prepared for making DNA mini preps
• designed primers for COMT gene (part of vanillin biosynthesis pathway)

Fri 20th July

• Prepared GFP and winter green biobricks for sequencing
• Revived Pseudamonas fluorescens from freeze dried culture (P. fluorescens contains two genes, ech and fcs, which are part of the synthetic vanillin biosynthesis pathway)
• Made 4 mini preps of ECFP
• Designed primers for ech and fcs genes
• Ordered Saccharothrix espeanensis from DSMZ culture collection, which contains Sam5 and Sam8 genes required for the synthetic vanillin biosynthesis pathway

Mon 23rd July

• Ran gels for ECFP mini preps

Restriction digested using XbaI and PstI

• Alfalfa growing well - now have enough leaf material from which to extract DNA

Tue 24th July

• Annealed dif F & R oligoes, restriction digested with SacI & SpeI, ligated with BioBrick vector Edinbrick 1

Wed 25th July

• Transformed dif F & R DNA into E.coli, incubated at 37C overnight
• designed primers for Sam5 and Sam8 genes (part of vanillin biosynthesis pathway)

Thu 26th July

• Prepared glycerol stock of Pseudomonas fluorescens
• Prepared miniprep of GFP and mint biobricks
• Streaked Pseudomonas fluorescens onto a fresh LB plate
• Revived [http://partsregistry.org/Part:BBa_I0500 BBa_I0500] and [http://partsregistry.org/Part:BBa_Q04510 BBa_Q04510]

Fri 27th July

• Continued miniprep of GFP and mint biobricks

Restriction digest using SpeI & EccRI restriction enzymes

Monday 30th July

• PCR of fcs and ech genes from Pseudomonas fluorescens was carried out

when pcr reaction was run on gel there was no product present, therefore we decided to carry out the pcr at a lower annealing temperature

Tuesday 31st July

• Prepared chloramphenicol plate to test if the revived wintergreen biobrick with the duel resistance plasmid was present
• Streaked out glycerol stock of Pseudomonas fluorescenes to determine if viable
• Carried out a second PCR of the ech and fcs genes required for vanillin biosynthesis pathway

lowered the annealing temperature to 54'C

• extracted DNA from alfalfa leaves ready for PCR amplification of COMT gene required for vanillin biosynthesis pathway

used the extract and amp kit provided by SIGMA

Wednesday 1st August

• PCR of ech and fcs was run on an agrose gel

results!! but not what we hoped for

fcs had several amplified fragments with one being present at the 1700bp mark which could represent the fcs gene

there was a vague band present at the 800bp mark, which possibly represents the ech gene

• decided to carry out pcr for a third time at a lower annealing temperature to suit the ech gene

fcs requires a higher annealing temperature to reduce the number of fragments amplifed

• streak plate of Pseudomonas fluorescens showed up positive

good news means our glycerol stock is viable :)

• chloramphenicol blue white selection plate came up positive for white colonies

shows that the transformed insert contains a plasmid with the chl resistance just like the wintergreen biobrick

Thursday 2nd August

• repeated ech PCR at a lower annealing temperature (reduced to 50'C)

gel showed that there was no improvement in the amplification of the ech gene

problem could be due to long extention time as ech PCR was ran at same time as a PCR for a larger gene

• PCR of COMT genes required for the vanillin biosynthesis pathway