1. The Goal
Put together the parts we have now (and some parts we’re still missing) to make:
LacI, TetR, and cI Lambda coding regions w/ hi/low constitutive promoters.
GFP reporters with pLac, pTet, and pLambda.
Put those two together on a p15a vector.
Prepare new vectors with Biobrick sites and Duet vector origin(s), like CloDF13 (P13).

2. How do we get there?
Hi/lo constitutive promoter + RBS + Repressor + Terminator
Lac/ Tet/ Lambda Promoter + RBS + GFP + Terminator

3. Hi/lo constitutive repressor: the plan…

4. …and what we have so far

5. More on Last Week’s Progress
We ligated RBS (P40) with TetR (P43) and cI Lambda (P44) coding regions to produce P Transformations were successful.
A Biobrick part with RBS + lacI + terminators was transformed and miniprepped last week (P54), so that is now also available.
P56-7 were digested, along with P38-9 (High/low constitutive promoters), and will be ligated and transformed today.
NEXT: Add terminator(s) (P41, 61-4). Then insert into p15a vector w/ GFP reporter.

6. Plan for p15A Vector w/ GFP Reporter

7. What do we have so far? Everything but…
Ligation of P45 + P18 happens today.

8. More on Last Week’s Progress
We electroporated and transformed Shewie MR-1 with P58 and P59, as well as P12 (pET-Duet) and some of Remy’s plasmids to see if the pSC101 origin works in S1.
P58 (Tet + GFP), as of Saturday, did not successfully transform.
P59 plate grew, and colonies were GREEN under the microscope. =)
Check those other plates!
NEXT: Grow liquid cultures and miniprep/ make glycerol stocks. Troubleshoot as to why P58 didn’t grow. Work on P74 and P75.

9. Making a New Vector w/ CloDF13 ori

10. Progress on CloDF13 vector?
We will ligate and transform modified P13 (CloDF13 ori) + P1 today.