IGEM:Harvard/2006/Perry projects

BioBricks assembly

The final constructs:

• R0010-B0032-Lpp29-OmpA66-Streptavidin

• R0010-B0032-Lpp29-OmpA159-Streptavidin

The parts

R0010 is a Lac promoter, B0032 is a ribosome binding site (medium strength, same one used in the GFP expression device), Lpp29-OmpA66 and Lpp29-OmpA159 are two versions of the membrane display scaffold.

There are several different versions of streptavidin that may be used. We have BioBricks of strepavidin clones from the Ting lab: wild-type, wild-type + His6tag, and dead, which I refer to as StrepW, StrepH, and StrepD.

Available sequenced and midiprepped parts

In Lpp-OmpA box...

• Lpp29, OmpA66, OmpA159 in pCR-2.1 Topo vector
• Lpp29, OmpA66, OmpA159 in pSB1A2 vector
• Lpp29-OmpA66 in pSB1A2

In StrepW,H,D box...

• StrepW, StrepH, StrepD in pCR-2.1 Topo vector

Available digested and gel purified parts, ready for ligation

• R0010, S/P (CIPed)
• Lpp29, E/S
• Lpp29, S/P (CIPed)
• OmpA66, X/P
• OmpA159, X/P
• Lpp29-OmpA66, S/P (CIPed)
• StrepW, StrepH, StrepD, X/P

• pSB1A2, E/P ("1")
• pSB1A2, E/S ("2")
• pSB1A2, X/P ("3")

BioBricks PCR

We're working on getting BioBricks of single-chain dimeric streptavidin clones from the Aslan lab: SCD-NM (single-chain dimer, not mutant), C2, and E2 (two mutant clones), which I refer to as S, C, and E. I just received a core streptavidin (streptavidin has a full-length version and a functional truncated core version) from the Dr. Takeshi Sano, called pTSA13, which I refer to as pTSA13 or just 13.

There are also two clones of the Lpp-OmpA scaffold which I received recently from the Georgiou lab, 481 (Lpp1-9-OmpA46-66-Bla) and 488 (Lpp1-9-OmpA46-145-Bla).

Goals for week of 8/6/06

Assembly

• Lpp29-OmpA159
• Lpp29-OmpA66-StrepW, H, or D
• R0010-B0032

BioBrick PCR

• SCD-NM, C2, or E2 clones of single-chain dimeric streptavidin from Aslan lab, with PstI site removed by mutagenesis
• pTSA13 streptavidin clone from Sano lab
• 481 or 488 construct of Lpp-OmpA from Georgiou lab

Agenda

Monday

• Inoculate liquid cultures, 3ml LB + 30ul amp(5mg/ml), of R0010 and B0032. You can use a R0010 streak on a carb plate labeled 7/31/06 in the B2 refrigerator, and a B0032 streak on a carb plate labeled 8/2/06. Make sure you start incubating/shaking them late enough in the day that you'll be around in 16 hours the following day to perform the miniprep.

• Prepare 25ul double digests using midipreps of Lpp29 (SpeI/PstI), OmpA159 (XbaI/PstI), and Lpp29-OmpA66 (SpeI/PstI) in pSB1A2 (Lpp-OmpA box). You should aim for around 1ug of DNA in each digest.
  • X ul midiprep for ~ug DNA, 2.5ul Buffer2, 0.5ul SpeI/XbaI, 0.5ul PstI, 0.25ul BSA(100X), Y ul water for total volume of 25ul
    • Y should equal 21.25 minus X. Don't worry about having exactly 0.25ul BSA; just convince yourself there's a little bit of BSA on the end of your pipet tip, and that it's entering your mixture.
  • Incubate at 37dC overnight (12h or so), and 80dC for 20min. There should be a program called "Digest" on either of the two grey PCR machines; just change the 37dC step to whatever time.

• Prepare three PCRs using "S/C/E no mut in pET" minipreps and StrepSCDF/StrepSCDRS(5uM) primers in "S,C,E" box, and PCR Supermix in "Primers/Stuff" box.
  • 45ul PCR Supermix + 2ul StrepSCDF(5uM) + 2ul StrepSCDRS(5uM) + 1ul "S, no mut in pET" miniprep
  • " + " + " + 1ul "C, no mut in pET" miniprep
  • " + " + " + 1ul "E, no mut in pET" miniprep

Tuesday

• Treat the digests with alkaline phosphatase (CIP).