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From 2011.igem.org

Contents 1 Monday 1.1 cI Repressor Test Assembly, Day 12 1.2 Cro and OR Insert into C3 Vector, Day 1 2 Tuesday 2.1 cI Repressor Test Assembly, Day 13 2.2 Cro and OR Insert into C3 Vector, Day 2 3 Wednesday 3.1 cI Repressor Test Assembly, Day 14 3.2 Cro and OR Insert into K3 Vector, Day 1 4 Thursday 4.1 cI Repressor Test Assembly, Day 15 4.2 Cro and OR Insert into K3 Vector, Day 1 5 Friday 6 =

Monday

cI Repressor Test Assembly, Day 12

The minipreps prepared on 6/4 (K648000+K648001, B0030+C0051, B0032+C0051) were sent to sequencing. The sequencing results showed that the K648000+K648001 and B0030+C0051 assemblies worked. However, the B0032+C0051 assembly only produced vector background. Thus, stock should be made of the K648000+K648001 and B0030+C0051 constructs and the B0032+C0051 assembly should be redone.

Cro and O_R Insert into C3 Vector, Day 1

The sensor group attempted to insert the Cro and O_R parts created through PCR on 6/3 into separate C3 vectors through amplified insert assembly.

Assembly Part	Part Involved with Insert Step
Restriction Digest	Inserts using XbaI and PstI:	OR Cro
	Vector using XbaI and PstI:	C3+violacein
Ligation	OR+C3 Cro+C3
Transformation/Plating	The above ligations were transformed into Escherichia coli cells and plated onto Chloramphenicol resistant plates.

Tuesday

cI Repressor Test Assembly, Day 13

The sensor group worked to make stock of their correct assemblies. The following constructs were transformed into Escherichia coli cells and allowed to grow in culture in order to make freezer stock.

K648000+K648001

B0030+C0051

B0034+C0051

Freezer stock was made from the B0031+C0051 culture as well.

Cro and O_R Insert into C3 Vector, Day 2

All of the colonies on the plates were purple, meaning that the insert never inserted into the vector. Thus, Cro and O_R must be inserted into a different vector.

Wednesday

cI Repressor Test Assembly, Day 14

The B0034+C0051 construct was sent to sequencing. The sequencing results showed that the assembly worked. Freezer stocks were made from the cultures made 6/7 (listed above).

Cro and O_R Insert into K3 Vector, Day 1

Since the C3 vector did not accept the Cro and O_R inserts, the sensor group attempted to insert Cro and O_R into the K3 vector.

Assembly Step	Part Involved with Assembly Step
Restriction Digest	Inserts using XbaI and PstI:	OR Cro
	Vector using XbaI and PstI:	K3
Ligation	OR+K3 Cro+K3
Transformation/Plating	The above ligations were transformed into Escherichia coli cells and plated onto Kanamycin resistant plates.

Thursday

cI Repressor Test Assembly, Day 15

The sensor group attempted to place a constitutive promoter (J23113) in front of the cI repressor and different ribosome binding sites.

Assembly Step	Part Involved with Assembly Step
Restriction Digest	Insert using EcoRI and SpeI:	J23113 B0033
	Vector using EcoRI and XbaI:	B0030+C0051 B0031+C0051 C0051
	Insert using XbaI and PstI:	C0051
	Vector using SpeI and PstI:	B0032
Ligation	J23113 + B0030+C0051 J23113 + B0031+C0051 B0033 + C0051 B0032 + C0051
Transformation/Plating	The above ligations were transformed into Escherichia coli cells and plated onto ampicillin resistant plates.

Cro and O_R Insert into K3 Vector, Day 1

K3 + OR colony A
K3 + OR colony B
K3 + Cro colony A
K3 + Cro colony B were all cultured in order to have the constructs sequenced.

Friday

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