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

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

• PCR of Pseudomonas fluorescens genes