"
Sensor: Week 2 May 23-27
From 2011.igem.org
(Difference between revisions)
(→Thursday) |
|||
| (12 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
==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+ | + | |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+ | + | |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+ | + | |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 | ||
| Line 67: | Line 78: | ||
|- | |- | ||
|Miniprep for<br />Sequencing | |Miniprep for<br />Sequencing | ||
| - | |style="color:green" colspan="2" align="center"|B0034+ | + | |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+ | + | |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+ | + | |B0034+mCherry+B0015 |
|- | |- | ||
|Vectors using EcoRI and XbaI: | |Vectors using EcoRI and XbaI: | ||
| Line 80: | Line 91: | ||
|- | |- | ||
|Ligation | |Ligation | ||
| - | |colspan="2" | B0034+ | + | |colspan="2" | B0034+mCherry+B0015 and J23113+B0034+C0051<br />B0034+mCherry+B0015 and J23105+B0034+C0051 |
|- | |- | ||
|Transformation/Plating | |Transformation/Plating | ||
| Line 87: | Line 98: | ||
<span style="color:green">Note:</span> The minipreps in green font indicate the sequencing proved the assembly worked. | <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
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
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
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 |
