Team:Evry/Notebook/Sensing/PCBs/08-20-2014

From 2014.igem.org

(Difference between revisions)
(Created page with "<html> <div class="cd-timeline-block"> </html> {{:Team:Evry/Notebook/Biology/TopImage}} <html> <div class="cd-timeline-content"> <p align="justify"> <u>Sensor constru...")
 
(6 intermediate revisions not shown)
Line 2: Line 2:
   <div class="cd-timeline-block">
   <div class="cd-timeline-block">
</html>
</html>
-
{{:Team:Evry/Notebook/Biology/TopImage}}
+
{{:Team:Evry/Notebook/Sensing/PCBs/TopImage}}
<html>
<html>
     <div class="cd-timeline-content">
     <div class="cd-timeline-content">
       <p align="justify">
       <p align="justify">
-
<u>Sensor construction hbpR2/PhbpR1</u>
+
<u>Sensor construction bphR2/PbphR1</u>
-
<br/>We received sequencing reads: that match perfectly to the registry sequence.
+
<br>We received sequencing reads: that match perfectly to the registry sequence.
-
<br/>26DJ54
+
<br>26DJ54
-
<br/>AATAGGCGTTATCACGAGGCAGAANTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGATGTCCCTGG
+
<br>AATAGGCGTTATCACGAGGCAGAANTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGATGTCCCTGG
GTGACATGCGTGATTTGGCCGCCACGCGGATCGCGCTCGAGAGCGAAGCGTTACGCCAAAGCGTGCTGAATGGTGACGCTGAATGGGAGGCGCGGATCGTCAGTTC
GTGACATGCGTGATTTGGCCGCCACGCGGATCGCGCTCGAGAGCGAAGCGTTACGCCAAAGCGTGCTGAATGGTGACGCTGAATGGGAGGCGCGGATCGTCAGTTC
GTTTCACCGACTGTCATTGATTGAAGAGCCCACGATGCGGGATCCGGCTCGCTGGTTTAATGAGTGGGAGCCAGTCAACCGCGGTTTTCACGAAGCTCTTATCTCT
GTTTCACCGACTGTCATTGATTGAAGAGCCCACGATGCGGGATCCGGCTCGCTGGTTTAATGAGTGGGAGCCAGTCAACCGCGGTTTTCACGAAGCTCTTATCTCT
Line 16: Line 16:
GGCTGCGGCGAGCGGTATCA
GGCTGCGGCGAGCGGTATCA
GCTCACTCAGGG
GCTCACTCAGGG
-
<br/>
+
<br>
-
<br/>26DJ55
+
<br>26DJ55
-
<br/>CGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGC
+
<br>CGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGC
AGCGGCCGCTACTAGTAAGCAGTCAATCGGCGGTAGCGCTCCATATGCACATACAGGATGGACAGGAACCGCCGGATCCAGACGGACGAACAGGCAGAGATAAGAG
AGCGGCCGCTACTAGTAAGCAGTCAATCGGCGGTAGCGCTCCATATGCACATACAGGATGGACAGGAACCGCCGGATCCAGACGGACGAACAGGCAGAGATAAGAG
CTTCGTGAAAACCGCGGTTGACTGGCTCCCACTCATTAAACCAGCGAGCCGGATCCCGCATCGTGGGCTCTTCAATCAATGACAGTCGGTGAAACGAACTGACGAT
CTTCGTGAAAACCGCGGTTGACTGGCTCCCACTCATTAAACCAGCGAGCCGGATCCCGCATCGTGGGCTCTTCAATCAATGACAGTCGGTGAAACGAACTGACGAT
Line 24: Line 24:
TCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA
TCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA
TGATAATAATGGTTTCTTAGA
TGATAATAATGGTTTCTTAGA
-
 
<br/>
<br/>
-
<br/><u>Sensor construction hbpR/PhbpC</u>
+
<br/>
-
<br/>The amplification by PCR was performed another time to amplify BBa_B0015 with overhangs. Protocol was the same as 08/19/2014. To verify PCR products, 10 µl was loaded on a 1% agarose gel with 2 µl of loading dye 6X. A really slight band was visible between 200 and 300 bp. It was to weak to obtain a picture with the camera. According to the ladder scale, the band correspond to 20-30 ng, so approximately 2-3 ng/µl of DNA for the PCR product.
+
<br/> Plasmid with bphR2 gene was purified with NucleoSpin Plamsid protocol (Macherey-Nagel) => we obtained 227,5ng/µL
 +
<br/>This plasmid was digested according this protocol:
 +
<ol>
 +
<li>Add:
 +
<ul>
 +
<li> sterilized water: qsp 20µL
 +
<li> template DNA : 500ng
 +
<li> buffer 2.1: 2µL
 +
<li> BSA: 0,2µL
 +
<li> EcoRI: 1µL
 +
<li> PstI: 1µL
 +
</ul>
 +
<li>Reverse for mix
 +
<li>Incubate at 37°C during 45mn
 +
<li>Incubate at 80°C during 20mn
 +
<li> Ligation was not possible because pSB1C3 plasmid was not digested so the digested plasmid (bphR2 gene) was stored at -20°C
 +
</ol>
 +
<br/>
 +
<u>Survival test on E.coli BL21:</u>
 +
<br/>Bacteria survive again for different concentrations but they were very concentrated
 +
<br/>
 +
<br/> A dilution of the medium has been done for the positive control and the six different concentrations:<br/>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2014/3/3a/2nd_Survival_test_1st_day.jpg" alt="image not found" /></div>
</p>
</p>

Latest revision as of 23:08, 17 October 2014

Picture

Sensor construction bphR2/PbphR1
We received sequencing reads: that match perfectly to the registry sequence.
26DJ54
AATAGGCGTTATCACGAGGCAGAANTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGATGTCCCTGG GTGACATGCGTGATTTGGCCGCCACGCGGATCGCGCTCGAGAGCGAAGCGTTACGCCAAAGCGTGCTGAATGGTGACGCTGAATGGGAGGCGCGGATCGTCAGTTC GTTTCACCGACTGTCATTGATTGAAGAGCCCACGATGCGGGATCCGGCTCGCTGGTTTAATGAGTGGGAGCCAGTCAACCGCGGTTTTCACGAAGCTCTTATCTCT GCCTGTTCGTCCGTCTGGATCCGGCGGTTCCTGTCCATCCTGTATGTGCATATGGAGCGCTACCGCCGATTGACTGCTTACTAGTAGCGGCCGCTGCAGTCCGGCA AAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC GGCTGCGGCGAGCGGTATCA GCTCACTCAGGG

26DJ55
CGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGC AGCGGCCGCTACTAGTAAGCAGTCAATCGGCGGTAGCGCTCCATATGCACATACAGGATGGACAGGAACCGCCGGATCCAGACGGACGAACAGGCAGAGATAAGAG CTTCGTGAAAACCGCGGTTGACTGGCTCCCACTCATTAAACCAGCGAGCCGGATCCCGCATCGTGGGCTCTTCAATCAATGACAGTCGGTGAAACGAACTGACGAT CCGCGCCTCCCATTCAGCGTCACCATTCAGCACGCTTTGGCGTAACGCTTCGCTCTCGAGCGCGATCCGCGTGGCGGCCAAATCACGCATGTCACCCAGGGACATC TCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA TGATAATAATGGTTTCTTAGA


Plasmid with bphR2 gene was purified with NucleoSpin Plamsid protocol (Macherey-Nagel) => we obtained 227,5ng/µL
This plasmid was digested according this protocol:

  1. Add:
    • sterilized water: qsp 20µL
    • template DNA : 500ng
    • buffer 2.1: 2µL
    • BSA: 0,2µL
    • EcoRI: 1µL
    • PstI: 1µL
  2. Reverse for mix
  3. Incubate at 37°C during 45mn
  4. Incubate at 80°C during 20mn
  5. Ligation was not possible because pSB1C3 plasmid was not digested so the digested plasmid (bphR2 gene) was stored at -20°C

Survival test on E.coli BL21:
Bacteria survive again for different concentrations but they were very concentrated

A dilution of the medium has been done for the positive control and the six different concentrations:
image not found

Aug 20

Retrieved from "http://2014.igem.org/Team:Evry/Notebook/Sensing/PCBs/08-20-2014"