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Work to be done:

Contents 1 Week of June 14/2010 1.1 Justin et al 1.1.1 Finish mms6-dT work 1.1.2 Test T4 DNA Ligase: 1.1.3 Prepare plasmid DNA for sequencing 1.1.4 Test PCR conditions for confirmation of ligation via PCR: 1.1.5 PCR Confirm previous ligations 1.2 Adam 2 Week of July 5th/2010 2.1 One 2.2 Two 2.3 Three 3 Week of July 12th/2010 3.1 One 3.2 Two 3.3 Three 4 Week of August 3rd/2010 4.1 One 4.2 Two 4.3 Three 5 Week of August 9th/2010

Week of June 14/2010

Justin et al

Finish mms6-dT work

• Heat kill ligase
• take small sample for gel
• take another sample for restriction (cut out entire biobrick)
• Run gel of above
• Transform into DH5α

Test T4 DNA Ligase:

• Restrict rbs-xylE with EcoRI and SpeI (take sample for analysis on gel)
• Heat kill EcoRI and SpeI
• Ligate with T4 DNA Ligase
• Run unrestricted, restricted and ligated samples on a gel.

Prepare plasmid DNA for sequencing

• Adam to upload guidelines for preparation.

Test PCR conditions for confirmation of ligation via PCR:

• Run a PCR of lumazine synthase gene that was sent for sequencing (and subsequently confirmed)
  • using VF2 and VR primers
• Run this PCR on a 2% agarose gel

PCR Confirm previous ligations

If (AND ONLY IF) PCR conditions amplified our known lumazine synthase gene, set up PCR of our previous ligation reactions to see if there is any ligated product.

Adam

• Finish VWR order (1000µL tips); test tubes; test tube racks; 96 well plates for sequencing, petri dishes
• Get RMA for VWR order (ie wrong test tube and test tube racks)
• Get guidelines for sequencing.
• Make primers for inserting mms6, xylE, lumazine, (C and N terminal labelled CFP/YFP-maybe) into pET28a expression vectors.No longer required.
• Order polymerase, SpeI, NotI, and (maybe, depending on results) T4 DNA ligase from Fermentas.
  • Check with HJ's lab to see which polymerase they use and from whom.
• Restrict lumazine, mms6, and xylE from BioBrick plasmid with NotI, and ligate into pET28a plasmid (pre-cut with NotI).
  • Check ligation and gel extract bands of proper size (Check for double insertion, plasmid self-ligation)
  • Transform into DH5α and purify plasmid DNA
  • Perform restriction analysis (using EcoRI and EcoRV) to check orientation of insert
  • Perform overexpression tests
  • Send for sequencing

Week of July 5th/2010

One

1) Maxiprep cells with the following BioBricks:
*pLacI
*pBad
*dT
*CFP compete
*Mms6 Mr. Gene
*pBad-TetR
*Lumazine
*EYFP

2) Request parts from the Registry

3) Send in for sequencing the products of PCR from June X/2010

4) Come up with PET28a plan B

5) Put flow chart on wall

Two

1) Add dT (from maxiprep above) to

• Mms6
• xylE
• Lumazine synthase

All above already maxiprepped
2)Overexpression test of "CFP Complete" and mms6

Three

• Restrict lumazine, mms6, and xylE from BioBrick plasmid with NotI, and ligate into pET28a plasmid (pre-cut with NotI).
  • Check ligation and gel extract bands of proper size (Check for double insertion, plasmid self-ligation)
  • Transform into DH5α and purify plasmid DNA
  • Perform restriction analysis (using EcoRI and EcoRV) to check orientation of insert
  • Perform overexpression tests
  • Send for sequencing

Week of July 12th/2010

One

1) Add dt to Mms6,Lumazine, and xylE

• Restrict Individual Parts
• Set up ligation reaction
• Confirm ligation using PCR
• Transform confirmed parts into DH5α
• Miniprep confirmed parts
  

2) Maxipreps

• pLacI
  

3) Jeff to finalize signal sequences by Wednesday for Friday order

4) Friday order for four fluorescent proteins

• CEYFP
• NEYFP
• CECFP
• NECFP
  

5) Successful meeting with Bob

6) Adam to talk to Lisza about Mms6

7) Adam to compile list of confirmed parts for Lisza

8) Transform Mms6 into BL21(DE3)

9) Justin/Mackenzie to talk to David about working with Argon to make nanoparticles

10) Send away all maxipreps for sequencing

11) Quantify maxipreps using gel method

12) Talk to Hayes lab about borrowing some Argon that they have on tap...

Two

1) maxiprep

• pET28(a)
  

2) Assemble

• pLacI-sRBS
  

Three

1) Assemble

• pLacI-sRBS-lumazine-dt
• pLacI-sRBS-xylE-dt
• pLacI-sRBS-Mms6-dt

2) Insert Mms6, xylE, and Lumazine Synthase into pET28(a) over-expression vector

Week of August 3rd/2010

One

1) Send samples for sequencing

• Registry Samples
• Miniprep samples
  

2) Talk to Nathan about PCR conditions for xylE

3) Justin to show other team members how to enter data into the wiki

4) Assemble

Two

Three

Week of August 9th/2010

1) PCR:

• Mms6 Mr. Gene with prefix/suffix
  
• xylE with xylE primers
• colonies with Phusion
• Fusion standards to fluorescent proteins
  

2) Prepare samples for sequencing

3) Prepare a list of parts for Lisza

4) Ligate Mms6 into pET-28(a)

5) Ligate lumazine into pET-28(a)

6) Assemble lumazine at dT

=Week of August 16th/2010=