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Reporter: Week 3 May 30- June 4
From 2011.igem.org
For week three, the reporter group continued creating the lac inducible test construct. When the primers, designed during week two, arrived, the reporters began assembling the linkers and cleavage sites via PCR.
Contents |
Monday, May 30
Lac Inducible Test Assembly: Take 2, Day 1
| Assembly Steps | Parts Involved with Assembly Step |
|---|---|
| PCR | J33204 miniprep from 5/26/11 |
| Restriction Digest | Insert: J33204 using XbaI and PstI Vector: R0010 using SpeI and PstI |
| Ligation | J33204+R0010 digests |
| Transformation/Plating | The above ligation was transformed into Escherichia coli cells and plated on Amp resistant plates. |
Tuesday, May 31
Lac Inducible Test Assembly: Take 2, Day 2
| Assembly Steps | Parts Involved with Assembly Step |
|---|---|
| Colony PCR | Two colonies from plates from 5/30/11 |
| Agarose Gel Elcectrophoresis | J33240 PCR products from 5/30/11 Colony PCR products from above |
Results: The gel showed that the insert (J33240) PCR worked, but the assembly did not work. We came to this conclusion because the constructs on the gel were less than 500 base pairs and our desired construct should have been about 1100 base pairs. Thus, we needed to do assembly for a third time. We started this third assembly with the R0100 miniprep from 5/26/11 and the J33204 PCR products from 5/30/11.
Lac Inducible Test Assembly: Take 3, Day 1
| Assembly Steps | Parts Involved with Assembly Step |
|---|---|
| Restriction Digest | Insert: J33204 using XbaI and PstI Vector: R0010 using SpeI and PstI |
| Ligation | J33204+R0010 digests |
| Transformation/Plating | The above ligation was transformed into Escherichia coli cells and plated onto Amp resistant plates. |
Wednesday, June 1
Lac Inducible Test Assembly: Take 3, Day 2
| Assembly Steps | Parts Involved with Assembly Step |
|---|---|
| Colony PCR | Two colonies from plates from 5/31/11 |
| Agarose Gel Elcectrophoresis | Colony PCR products from above |
Results:The gel showed that the assembly did not work. The gel had the same results of the gel from last week and the one from 5/31/11. We determined that we need to use a more specific ratio of insert DNA to vector DNA. We will determine this ratio in our next assembly. The fourth assembly began today with the transformation of J33204 and R0010 from the registry into Escherichia coli cells.
Thursday, June 2
Lac Inducible Test Assembly: Take 4, Day 1
| Assembly Step | Parts Involved with Assembly Step |
|---|---|
| Miniprep | J33204 R0010 |
| PCR | J33204 |
| Nanodrop* | J33204 R0010 |
| Restriction Digest** | Insert: J33204 using XbaI and PstI Vector: R0010 using SpeI and PstI |
| Ligation*** | 4:1 ratio of insert to vector 6:1 ratio of insert to vector |
| Transformation/Plating | The above ligation was transformed into Escherichia coli cells and plated onto Amp resistant plates. |
*Note:We needed to determine the concentration of DNA in our miniprep products, so we used a nanodrop. Our results are shown below.
| Registry Part | Concentration (ng/μl) | 260/280 |
|---|---|---|
| J33240 | 22.9 | 1.93 |
| R0010 | 137.3 | 1.90 |
We used this information to calculate the concentration (in picomoles/microliter) of our insert and vector DNA. The calculation we used was:
(concentration of DNA)/[(0.66)*(size of part in base pairs)]
We wanted 0.25 pM of vector and 0.50 pM of insert, so we calculated how much volume of the insert and vector digests we needed to add for our ligation. These volumes as well as the molar concentrations of our minipreps are summarized in the below table.
| Registry Part | Molar Concentration (pM/μl) | Volume for Ligation (μl) |
|---|---|---|
| J33204 | 0.0119 | 21.0 |
| R0010 | 0.223 | 2.24 |
**Note:The following changes were made from the insert digest protocol:
| dH20: | 39.26 μl |
| J33204: | 2.24 μl |
The following changes were made from the vector digest protocol:
| dH20: | 21.0 μl |
| R0010: | 21.0 μl |
***Note:To get a 4:1 ratio of insert DNA to vector DNA, the following changes were made to the ligation protocol:
| Insert (J33240): | 2 μl |
| Vector (R0010): | 4 μl |
To get a 6:1 ratio of insert DNA to vector DNA, the following changes were made to the ligation protocol:
| Insert (J33204): | 1.5 μl |
| Vector (R0010): | 4.5 μl |
Friday, June 3
Lac Inducible Test Assembly: Take 4, Day 2
| Assembly Step | Parts Involved with Assembly Step |
|---|---|
| Colony PCR | 4:1 ratio colonies 6:1 ratio colonies |
| Agarose Gel Electrophoresis | 4:1 colony PCR products 6:1 colony PCR products |
| Culture | The 6:1 ratio colony that worked based on the gel electrophoresis was grown overnight. |
Gel Results: One of the 6:1 ratio constructs contained the correct amount of base pairs (about 1100). The other constructs did not have more than 500 base pairs. The 6:1 ratio construct will be sequenced to determine its legitimacy.
PCR Assembly, Day 1
Now that our primers, which were designed last week, arrived, we started constructing the linkers and cleavage sites via PCR. The registry names and our colloquial names (names we refer to these parts as throughout this notebook) are summarized in the following table. We followed the Assembly via PCR protocol for these assemblies.
| Registry Name | Colloquial Name |
|---|---|
| K243004 | small linker |
| K105012 | 10AA linker |
| K316007 | Imperial linker |
| N/A | tev cleavage site |
| N/A | cI cleavage site |
- Note: When purifying the PCR products, DNA wash buffer without ethanol was used, making all of the PCR products unusable. These assemblies will have to be redone tomorrow.
Saturday, June 4
Lac Inducible Test Assembly: Take 4, Day 3
The Plac+XylE (6:1 ratio) culture was miniprepped for sequencing.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Miniprep | J33240+R0010 |
PCR Assembly, Day 2
The PCR reactions from 6/3/11 were run on a 1% agarose gel to see if the assembly worked. The PCR products were purified.
| Assembly Step | Part Involved with Assembly Step |
|---|---|
| Agarose Gel Electrophoresis | Imerial linker cI cleavage site tev cleavage site 10AA linker small linker |
| PCR Purification | Imerial linker cI cleavage site tev cleavage site 10AA linker small linker |
*Results: The agarose gel electrophoresis showed that the all of the PCR assembled small parts contain the correct number of basepairs.
Insert Imperial Linker into C3 Vector, Day 1
The Imperial Linker was inserted into the violacein vector (C3) via amplified insert assembly. Then, the Imperial Linker can be extracted from the C3 vector so other small parts can be inserted into the C3 vector. This way we can keep the small parts in a plasmid.
| Assembly Step | Part Involved with Assembly Step | |
|---|---|---|
| Restriction Digest | Insert using XbaI and PstxI | Imperial linker |
| Vector using XbaI and PstxI | Violacein vector | |
| Ligation | Imperial linker+C3 | |
| Transformation/Plating | The above ligation was transformed into Escherichia coli cells and plated onto chloramphenicol resistant plates. | |
