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From 2011.igem.org

Contents 1 Monday 1.1 Lac Inducible Test Assembly: Take 4, Day 4 1.2 Insert Imperial Linker into C3 Vector, Day 2 1.3 Extract LacZα from stock part, Day 1 2 Tuesday 2.1 Lac Inducible Test Assembly: Take 4, Day 5 2.2 Insert Imperial Linker into C3 Vector, Day 3 3 Wednesday 3.1 Lac Inducible Test Assembly: Take 4, Day 6 3.2 Extract tev, GFP, XylE from Registry Parts, Day 1 3.3 Insert Imperial Linker into K3 Vector, Day 1

Monday

Lac Inducible Test Assembly: Take 4, Day 4

The reporter group took the miniprep for the P_lac+XylE construct to sequencing. The sequence results showed that the P_lac assembled with the first 750 bp of XylE. Thus, the assembly was verified.

Insert Imperial Linker into C3 Vector, Day 2

The reporter group checked to see if the insertion of the Imperial linker into the C3 vector worked.

Assembly Step	Part Involved with Assembly Step
Colony PCR	Imperial linker+C3
Agarose Gel Electrophoresis	The colony PCR product was run on a 1% agarose gel.
Culture	5 ml of SOB media with 6 μl of chloramphenicol was innoculated with the Imperial linker+C3 colony.

• Results: The gel showed a band less than 100 base pairs which matches the Imperial linker's 85 base pair length.

Extract LacZα from stock part, Day 1

The reporter group ran PCR to knock out the LacZα part from Mike's stock. The agarose gel showed that the PCR product contained the correct number of base pairs.

Tuesday

Lac Inducible Test Assembly: Take 4, Day 5

The reporter group began making stock of the P_lac+XylE.

Assembly Step	Part Involved with Assembly
Transformation	J33204+R0010 was transformed into Escherichia coli cells.
Culture	The SOB media and the Amp antibiotic mixture was innoculated with the transformed cells from above. The culture was left shaking overnight.

Insert Imperial Linker into C3 Vector, Day 3

The Imperial linker+C3 culture was miniprepped and taken to sequencing. The results showed the Imperial linker with a truncated prefix (no XbaI), followed by violacein. Thus, the assembly failed.

Wednesday

Lac Inducible Test Assembly: Take 4, Day 6

The reporter group prepared freezer stock of the correctly assembled P_lac+XylE construct, as well as the following parts from the registry.

Registry Part	Colloquial Name
K327018	cI+LVA tag
K082009	Lambda repressor
K316007	GFP-XylE Imperial part (for testing)
K316017	Lac controlled tev
J33204+R0010	Plac+XylE with RBS

Extract tev, GFP, XylE from Registry Parts, Day 1

The reporter group performed PCR to extract tev, GFP and XylE from their registry parts. The specific parts are listed below:

Gene of Interest	Registry Part	Length of Gene of Interest (bp)
K316007:	XylE	925
K316007:	GFP+flag tag	800
K316017:	tev protease	720

Assembly Step	Part Involved with Assembly Step
Miniprep	K316007 K316017
PCR	K316007 (twice with different primers, designed May 24) K316017
Agarose Gel Electrophoresis	All above PCR products

• Results: The gel showed bands all corresponding to 1000 base pairs, which is approximately correct for the genes of interest. Sequencing will further verify our PCR products.

Insert Imperial Linker into K3 Vector, Day 1

Since inserting the Imperial linker into the C3 vector did not work, we tried inserting the Imperial linker into the K3 vector, using the purified PCR product from 6/4/11. We did the insertion through amplified insert assembly.

Assembly Step	Part Involved with Assembly Step
Restriction Digest	Insert using XbaI and PstI:	Imperial Linker
	Vector using XbaI and PstI:	K3
Ligation	Imperial linker+K3
Transformation/Plating	The above ligation was transformed into Escherichia coli cells and plated onto Kanamycin resistant plates to let grow overnight.

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