IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week2/Chemical and Light

=Goals for Week 2=
 * 1) Transform LacI, TetR, cI + their promoters and GFP. Refer [here].
 * 2) Cut Lac promoter -> GFP (P20), and Tet promoter -> GFP (P15), with Xba and PstI and cut SB3K3 (P5) with Xba and PstI and ligate.
 * 3) Transform cI promoter. (Refer [here].
 * 4) Put constitutive promoters on TetR, LacI, cI.
 * 5) Put terminators and RBS on TetR, LacI, and cI.
 * 6) Add promoters.
 * 7) Make primers that have BioBricks prefix and suffix to flank the origin on Duet Vectors so we can take it out.
 * 8) PCR out mtrA and mtrB from Shewie. Also put in with repressors.
 * 9) Test anaerobic growth further with birnesite.

=Ongoing Experiments=

Sequencing and PCRs

 * 6/30: Primers to PCR out the CDF origin of replication (with BioBrick ends) were ordered.
 * 7/1: PCR reaction set up: 45μL PCR supermix, 1μL S1P13 (435ng/μL), 1μL CDF-F primer (20μM), 1μL CDF-R primer (20μM), 2μL water. Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 58°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid
 * 6/30: Most of the minipreps of BioBricks that have been done have been prepared to be sent for sequencing. For longer sequences, both the BBsfx and the BBpfx primers were used. For shorter sequences, only the BBsfx primer was used (this primer is longer and therefore gives more leeway for garbled bases at the beginning of a read.)
 * 6/30: 5mL LB-Kan culture of P1 is growing (from glycerol stock)- the plasmid can be digested to yield pSB3K3

=The Plan=

Goal: High/ Low Constitutive Promoter + RBS + Repressor Coding Region for cI Lambda/ TetR/ LacI + Terminator(s) + pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori

New Vector w/ CDF ori + Resistance Marker
=Transformations from the Registry=

Re-Transformation of BioBrick Parts in E1 and E3 (6/27-7/1)
Miniprepped and made glycerol stocks of Biobrick Parts transformed on Friday.

Transformation of Terminators and LacI coding regions (7/2)
=First Round= Putting Lac/Tet + GFP with P15A, and RBS with TetR and cI lambda coding regions.

In this round:

Also cutting P38, P39, and P45 in anticipation of future ligations.

Restriction Enzyme Digestions (7/1)
Vectors (digested with SpeI and PstI)
 * RBS (P40, P45)
 * cI regulated lambda promoter (P18)
 * promoters (P38, P39)

Inserts (digested with XbaI and PstI)
 * repressor coding regions (P43, P44)
 * GFP under Tet promoter (P15)
 * GFP under Lac promoter (P20)
 * vector with p15A origin (P1)

Reaction mixture
 * 15 μl DNA
 * 6.25 μl H2O
 * 2.5 μl 10X NEB buffer (buffer 3 for XP, buffer 2 for SP)
 * 0.5 μl 50X BSA
 * 0.5 μl each RE

Reactions:

Incubate overnight at 37 °C

Gel Extraction and Purification of First Round Digestions (7/1)


Gel 1

Gel 2

Ligation and Transformation (7/1)
We are ligated the repressor coding regions (P43 and P44) to the RBS (P40). We are also going to put P15 and 20 into a p15A vector (P1). After sequencing P18 (cI regulated lambda promoter) we will add it to P45 (RBS with GFP).

We transformed DH5α cells with each of these new plasmids (P40+43, P40+44, P1+15, P1+20).

Picking Colonies (7/2)
All four transformations were successful, with many colonies on each plate. Two colonies were picked from each plate.

Transformation of P58 and P59, and pET-Duet-1 into Shewanella via Electroporation (7/3)
=Second Round=  - Adding hi/low constitutive promoters to repressors, OmpR-P promoter to p15a, and p-lambda to (RBS + GFP)

(Projected Third Round)

(Projected Fourth Round)

Digestions (7/3)
Digestion Reactions:

First Batch

Second Batch (We did some of the digestions in the First Batch incorrectly. Only the correct ones are now listed under First Batch.) All of these parts were run on a gel and purified

=Transformation of Parts into S1=

Transformation of Lac/Tet + GFP into S1(7/3)
25 mL culture of S1 was grown for electroporation and transformation.