Difference between revisions of "Team:Goettingen/Results"
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<p>ESTERASE AND PHOSPHATASE CONSTRUCTS</p> | <p>ESTERASE AND PHOSPHATASE CONSTRUCTS</p> | ||
<p>Esterase was amplified from pET101_E064 and phosphatase from pTOPOXL_PLP07 by PCR for each enzyme (Fig.1). Both plasmids were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites <em>Kpn</em>I and <em>Sac</em>I for esterase and <em>Xho</em>I and <em>Pst</em>I for the phosphatase in order to make them compatible for insertion into the multiple cloning site of the pBAD vector .The genes for these two enzymes had been found in screenings of metagenomic libraries.</p> | <p>Esterase was amplified from pET101_E064 and phosphatase from pTOPOXL_PLP07 by PCR for each enzyme (Fig.1). Both plasmids were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites <em>Kpn</em>I and <em>Sac</em>I for esterase and <em>Xho</em>I and <em>Pst</em>I for the phosphatase in order to make them compatible for insertion into the multiple cloning site of the pBAD vector .The genes for these two enzymes had been found in screenings of metagenomic libraries.</p> | ||
| + | <img src=" https://static.igem.org/mediawiki/2015/1/15/PCR_esterase_phosphatase_iGEM_Team_Goettingen_2015.jpeg | ||
| + | " style="; float:right;"> | ||
<p>After purification of the PCR products, they were ligated into pJET by blunt end ligation. This vector serves to clone the enzymes without triggering their activity, which may interact with the vector or the <em>E.coli</em>. In the case of phosphatase, the PCR products needed to be purified by gel extraction, to eliminate left over template plasmids, which could interfere with the transformation.</p> | <p>After purification of the PCR products, they were ligated into pJET by blunt end ligation. This vector serves to clone the enzymes without triggering their activity, which may interact with the vector or the <em>E.coli</em>. In the case of phosphatase, the PCR products needed to be purified by gel extraction, to eliminate left over template plasmids, which could interfere with the transformation.</p> | ||
<p>Ligation into pJET was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with a Quiaprep spin Miniprep kit and restricted with either <em>Kpn</em>I and <em>Sac</em>I or <em>Xho</em>I and <em>Pst</em>I, depending on the Enzyme (Fig.2&3).</p> | <p>Ligation into pJET was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with a Quiaprep spin Miniprep kit and restricted with either <em>Kpn</em>I and <em>Sac</em>I or <em>Xho</em>I and <em>Pst</em>I, depending on the Enzyme (Fig.2&3).</p> | ||
Revision as of 08:17, 16 September 2015
Project Results
Transformation Efficiency Kit, RFP construct (iGEM)
Before using our competent E. coli TOP10 cells in the important experiments, we used the Transformation Efficiency Kit to test the efficiency of our competent cells!
The kit includes five vials of each different DNA concentration: 50pg/μl, 20pg/μl, 10pg/μl, 5pg/μl, 0.5pg/μl of purified DNA from BBa_J04450 (RFP construct) in plasmid backbone pSB1C3.
The first test transformation (50 μl of competent cells, 20 μl plated) showed very poor results:
|
DNA concentration |
0.5pg/μl |
5pg/μl |
10pg/μl |
20pg/μl |
50pg/μl |
|
# of colonies |
0 |
0 |
2 |
0 |
13 |
|
efficiency (cfu) |
0 |
0 |
3.8x106 |
0 |
3.4x106 |
|
Results |
efficiency |
|
average |
1.4x106 |
|
weighted |
3.5x106 |
The second test transformation (200 μl of competent cells, 100 μl plated) showed still poor results but we decided to continue working with our cells:
|
DNA concentration |
0.5pg/μl |
5pg/μl |
10pg/μl |
20pg/μl |
50pg/μl |
|
# of colonies |
1 |
13 |
1 |
35 |
28 |
|
efficiency (cfu) |
8.0x106 |
1.0x107 |
4.0x105 |
7.0x106 |
2.3x106 |
|
Results |
efficiency |
|
average |
5.6x106 |
|
weighted |
3.7x106 |
RFP construct
ESTERASE AND PHOSPHATASE CONSTRUCTS
Esterase was amplified from pET101_E064 and phosphatase from pTOPOXL_PLP07 by PCR for each enzyme (Fig.1). Both plasmids were given to us by the Applied and Genomic Microbiology department. Primers contained restriction sites KpnI and SacI for esterase and XhoI and PstI for the phosphatase in order to make them compatible for insertion into the multiple cloning site of the pBAD vector .The genes for these two enzymes had been found in screenings of metagenomic libraries.
After purification of the PCR products, they were ligated into pJET by blunt end ligation. This vector serves to clone the enzymes without triggering their activity, which may interact with the vector or the E.coli. In the case of phosphatase, the PCR products needed to be purified by gel extraction, to eliminate left over template plasmids, which could interfere with the transformation.
Ligation into pJET was followed according to the protocol in the methods collection. After over-night incubation colonies were picked, plasmids extracted with a Quiaprep spin Miniprep kit and restricted with either KpnI and SacI or XhoI and PstI, depending on the Enzyme (Fig.2&3).
Once restrictions showed the correct bands, both pJET containing esterase and phosphatase were sent for sequencing by the G2L laboratory.
Esterase and Phosphatase construct
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