Sensor: Week 2 May 23-27

From 2011.igem.org

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==Monday==
==Monday==
 +
===cI Repressor Test Assembly, Day 1===
 +
The sensor group began the assembly of a cI repressor testing construct by amplifying the below constitutive promoters via PCR.
{|border="1"
{|border="1"
!Assembly Step
!Assembly Step
Line 9: Line 11:
==Tuesday==
==Tuesday==
 +
===cI Repressor Test Assembly, Day 2===
 +
[[File:Jamie_research.jpg|thumb|right|400px|Jamie says: "No more research. To the lab!"]]
 +
The sensor group continued to build their test construct by inserting the constitutive promoters into the vector containing the cI repressor and ribosome binding site (B0034+C0051). The sensor group also inserted a ribosome binding site and color reporter (B0034+mCherry) in front of a terminator (B0015).
{|border="1"
{|border="1"
|'''Assembly Step'''
|'''Assembly Step'''
Line 21: Line 26:
|rowspan="2" | Restriction Digest
|rowspan="2" | Restriction Digest
|Inserts using EcoRI and SpeI:
|Inserts using EcoRI and SpeI:
-
|J23113<br />J23105<br />J23118<br />J23100<br />B0034+Mcherry
+
|J23113<br />J23105<br />J23118<br />J23100<br />B0034+mCherry
|-
|-
|Vectors using EcoRI and XbaI:
|Vectors using EcoRI and XbaI:
Line 27: Line 32:
|-
|-
|Ligation
|Ligation
-
|colspan="2" align="center" | J23113 and B0034+C0051<br />J23105 and B0034+C0051<br />J23118 and B0034+C0051<br />J23100 and B0034+C0051<br />B0034+Mcherry and B0015
+
|colspan="2" align="center" | J23113 and B0034+C0051<br />J23105 and B0034+C0051<br />J23118 and B0034+C0051<br />J23100 and B0034+C0051<br />B0034+mCherry and B0015
|-
|-
|Transformation/Plating
|Transformation/Plating
Line 37: Line 42:
==Wednesday==
==Wednesday==
 +
===cI Repressor Test Assembly, Day 3===
 +
[[File:Everyone_lab_waiting_safe.jpg|right|thumb|400px|Jamie says: "D'oh! No one said anything about WAITING!"]]
 +
The sensor group inserted various ribosome binding sites (listed below) in front of the cI repressor (C0051). The sensor group also finished the assemblies from yesterday by performing colony PCR on the plated colonies.
{|border="1"
{|border="1"
!Assembly Step
!Assembly Step
Line 42: Line 50:
|-
|-
|Colony PCR
|Colony PCR
-
|colspan="2" | B0034+Mcherry+B0015 (Kan resistance)<br />B0034+Mcherry+B0015 (Amp resistance)
+
|colspan="2" | B0034+mCherry+B0015 (Kan resistance)<br />B0034+mCherry+B0015 (Amp resistance)
|-
|-
|PCR Purification
|PCR Purification
Line 62: Line 70:
==Thursday==
==Thursday==
 +
===cI Repressor Test Assembly, Day 4===
 +
[[File:Oligos_arrival.jpg|thumb|right|350px|Finally, the oligos arrived!]]
 +
The sensor group put the reporting assembly (B0034+mCherry+B0015) in front of two cI repressor construct with different constitutive promoters (J23113+B0034+C0051 and J23105+B0034+C0051).
{|border="1"
{|border="1"
!Assembly Step
!Assembly Step
-
|colspan="2" align="center"|Part Involved with Assembly Step
+
|colspan="2" align="center"|'''Part Involved with Assembly Step'''
|-
|-
|Miniprep for<br />Sequencing
|Miniprep for<br />Sequencing
-
|style="color:green" colspan="2" align="center"|B0034+Mcherry+B0015<br />J23113+B0034+C0051<br />J23105+B0034+C0051
+
|style="color:green" colspan="2" align="center"|B0034+mCherry+B0015<br />J23113+B0034+C0051<br />J23105+B0034+C0051
|-
|-
|PCR
|PCR
-
|colspan="2" align="center"|B0034+Mcherry+B0015
+
|colspan="2" align="center"|B0034+mCherry+B0015
|-
|-
|rowspan="2"|Restriction Digest
|rowspan="2"|Restriction Digest
|Insert using EcoRI and SpeI:
|Insert using EcoRI and SpeI:
-
|B0034+Mcherry+B0015
+
|B0034+mCherry+B0015
|-
|-
|Vectors using EcoRI and XbaI:
|Vectors using EcoRI and XbaI:
Line 80: Line 91:
|-
|-
|Ligation
|Ligation
-
|colspan="2" | B0034+Mcherry+B0015 and J23113+B0034+C0051<br />B0034+Mcherry+B0015 and J23105+B0034+C0051
+
|colspan="2" | B0034+mCherry+B0015 and J23113+B0034+C0051<br />B0034+mCherry+B0015 and J23105+B0034+C0051
|-
|-
|Transformation/Plating
|Transformation/Plating
|colspan="2" | The above ligations were transformed into Escherichia<br />coli cells and plated onto Amp resistant plates.
|colspan="2" | The above ligations were transformed into Escherichia<br />coli cells and plated onto Amp resistant plates.
|}
|}
 +
<span style="color:green">Note:</span> The minipreps in green font indicate the sequencing proved the assembly worked.
 +
==Friday==
 +
===cI Repressor Test Assembly, Day 5===
 +
The week's assemblies have been confirmed and named for the registry.
 +
{|border="1"
 +
!Assembly
 +
!Registry Name
 +
|-
 +
|B0034+mCherry+B0015<br />J23113+B0034+C0051 <br />J23105+B0034+C0051
 +
|style="color:purple" align="right" | K648000<br />K648001<br />K648002
 +
|}

Latest revision as of 15:35, 10 June 2011

Contents

Monday

cI Repressor Test Assembly, Day 1

The sensor group began the assembly of a cI repressor testing construct by amplifying the below constitutive promoters via PCR.

Assembly Step Parts Involved in
Assembly Step
PCR J23113
J23105
J23118
J23100

Tuesday

cI Repressor Test Assembly, Day 2

Jamie says: "No more research. To the lab!"

The sensor group continued to build their test construct by inserting the constitutive promoters into the vector containing the cI repressor and ribosome binding site (B0034+C0051). The sensor group also inserted a ribosome binding site and color reporter (B0034+mCherry) in front of a terminator (B0015).

Assembly Step Parts Involved in Assembly Step
PCR Purification J23113
J23105
J23118
J23100
Miniprep B0051
Restriction Digest Inserts using EcoRI and SpeI: J23113
J23105
J23118
J23100
B0034+mCherry
Vectors using EcoRI and XbaI: B0034+C0051
B0015
Ligation J23113 and B0034+C0051
J23105 and B0034+C0051
J23118 and B0034+C0051
J23100 and B0034+C0051
B0034+mCherry and B0015
Transformation/Plating The above ligations were transformed into Escherichia
coli cells and plated onto Amp resistant plates.
PCR B0030
B0031
B0032
B0033

Wednesday

cI Repressor Test Assembly, Day 3

Jamie says: "D'oh! No one said anything about WAITING!"

The sensor group inserted various ribosome binding sites (listed below) in front of the cI repressor (C0051). The sensor group also finished the assemblies from yesterday by performing colony PCR on the plated colonies.

Assembly Step Parts Involved with Assembly Step
Colony PCR B0034+mCherry+B0015 (Kan resistance)
B0034+mCherry+B0015 (Amp resistance)
PCR Purification B0030
B0031
B0032
B0033
Restriction Digest Inserts using EcoRI and SpeI: B0030
B0031
B0032
B0033
Vector using EcoRI and XbaI: C0051
Ligation B0030+C0051
B0031+C0051
B0032+C0051
B0033+C0051
Transformation/Plating The above ligations were transformed
into Escherichia coli cells and plated onto
Amp resistant plates.

Thursday

cI Repressor Test Assembly, Day 4

Finally, the oligos arrived!

The sensor group put the reporting assembly (B0034+mCherry+B0015) in front of two cI repressor construct with different constitutive promoters (J23113+B0034+C0051 and J23105+B0034+C0051).

Assembly Step Part Involved with Assembly Step
Miniprep for
Sequencing 		B0034+mCherry+B0015
J23113+B0034+C0051
J23105+B0034+C0051
PCR B0034+mCherry+B0015
Restriction Digest Insert using EcoRI and SpeI: B0034+mCherry+B0015
Vectors using EcoRI and XbaI: J23113+B0034+C0051
J23105+B0034+C0051
Ligation B0034+mCherry+B0015 and J23113+B0034+C0051
B0034+mCherry+B0015 and J23105+B0034+C0051
Transformation/Plating The above ligations were transformed into Escherichia
coli cells and plated onto Amp resistant plates.

Note: The minipreps in green font indicate the sequencing proved the assembly worked.

Friday

cI Repressor Test Assembly, Day 5

The week's assemblies have been confirmed and named for the registry.

Assembly Registry Name
B0034+mCherry+B0015
J23113+B0034+C0051
J23105+B0034+C0051 		K648000
K648001
K648002





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