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Team:INSA Toulouse/contenu/lab practice/notebook/calendar/logic gates
From 2013.igem.org
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<div class="infos"> | <div class="infos"> | ||
<ul class="circlearrow"> | <ul class="circlearrow"> | ||
| + | <li><span class="spantitle2">Week 4 (1-7 July)</span><br> | ||
| + | 04/07<br> | ||
| + | Summary of all the work we have to do for the logic gates. These are the necessary genes we needed to cloned into the logic gates.<br> | ||
| + | Fro And1: rbs-luxI and rbs-Ci-ter<br> | ||
| + | For Xor1: rbs FimE ter<br> | ||
| + | For And2: reversed rbs luxI<br> | ||
| + | For Xor2: reversed rbs-RFP-ter<br><br> | ||
| + | </li> | ||
| + | |||
<li><span class="spantitle2">Week 6 (15-21 July)</span><br> | <li><span class="spantitle2">Week 6 (15-21 July)</span><br> | ||
| - | + | Amplification of fimE recombinase (rbs+fimE) from the Michigan Team.<br> | |
| - | + | Minipreps, and electrophoresis checking-> good amplification.<br><br> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
</li> | </li> | ||
| - | + | ||
| - | + | <li><span class="spantitle2">Week 7 (22-28 July)</span><br> | |
| - | + | Problem with AND1, the synthesis is not correct. We cannot go on with this gate. We planned order a new synthesis.<br> | |
| + | Amplification of a new rbs-luxI (BBa_K516011). We realized that the first rbs –luxI we amplified was not the good one (wrong size).<br> | ||
| + | Cloning of rbs+fimE with a terminator, wrong electrophoresis profile.<br> | ||
| + | Cloning of rbs-luxI with rbs-CI-ter. This cloning step was successful.<br> | ||
| + | Transfer of xor2 synthesis from puc54 to pSB1C3. We did not get xor2 inside pSB1C3, but only the backbone vector.<br> | ||
| + | Insertion of reversed RFP inside AND2 (with cla1/bamH1). Electrophoresis profiles were not correct, so the insertion was not successful. It was probably a problem of restriction time. | ||
| + | <br><br> | ||
</li> | </li> | ||
</ul></li> | </ul></li> | ||
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<div class="infos"> | <div class="infos"> | ||
<ul class="circlearrow"> | <ul class="circlearrow"> | ||
| - | <li><a href="">Week | + | <li><a href="">Week 8 (29-04 August)</a><br> |
| - | <li><a href="">Week | + | Cloning of rbs+fimE with a terminator. This time we succeeded.<br> |
| + | Amplification of the fragment rbs luxI-rbs-CI-ter for the AND1 gate.<br> | ||
| + | Insertion of rbs-FImE-ter inside the Xor1 gate. We made restriction controls with different enzymes (stu1,hindIII), but the results were not fruitful.<br> | ||
| + | We tried again to introduce RFP inside AND2, still not working.<br> | ||
| + | We succeeded to transfer Xor2 inside pSB1C3.<br> | ||
| + | </li> | ||
| + | <li><a href="">Week 9 (05-11 August)</a><br> | ||
| + | Still lot of problems to insert reversed RFP inside AND2. The transformation does not work. We needed to find a solution (strataclone? Infusion?...).<br> | ||
| + | Insertion of reversed rbs luxi inside AND2. Same problem as for AND2-RFP.<br> | ||
| + | Cloning of rbs-fimE-ter into Xor1. It did not work because of a ligation problem.<br> | ||
| + | Insertion of reversed RFP into XOR2, no insertion, only the backbone vector was present.<br> | ||
| + | </li> | ||
| + | <li><a href="">Week 10 (12-18 August)</a><br> | ||
| + | As we faced lot of issues with the reversed RFP and reversed rbs-luxI, we decided to order new oligonucleotide in order to create new reversed fragments as we did during the week 6 | ||
| + | PCR to invert RFP and rbs.LuxI with the restriction site ClaI BamHI, and purification.<br> | ||
| + | We tested a wide range of elongation temperatures (60°C-75°C). We got the right fragments for the two biobricks with good size. But on the electrophoresis revelations we got one parasite fragment around 5kb. | ||
| + | <br> | ||
| + | </li> | ||
| + | <li><a href="">Week 11 (19-25 August)</a><br> | ||
| + | We amplified the reversed fragment RFP and rbs luxI with the new oligos. We did not find the solution to avoid the parasite fragment so we used PCR clean-up & gel purification.<br> | ||
| + | Insertion of these fragments into the gate (rbs-luxI into AND2 and RFP into XOR2 and AND2).<br> We did not get better results even with these new fragments.<br><br> | ||
| + | We realized in fact we have a problem with the XOR2 gate. Instead of having only one site cla1 inside the XOR2, we had two. The problem was therefore the restriction with cla1!<br><br> | ||
| + | Extraction And 1 gate from And gate part of Bonnet construction by PCR.<br> | ||
| + | Cloning : (Digestion, Ligation, Transformation , Pre-culture, Miniprep )<br> | ||
| + | Insertion of And 1 gate extracted by PCR into the plasmid backbone pSB1K3<br> | ||
| + | Assembly of And 1 gate extracted by PCR and rbs RFP term part<br> | ||
| + | Assembly of Xor 1 gate and rbs RFP term parts<br> | ||
| + | <br> | ||
| + | </li> | ||
| + | |||
</ul> | </ul> | ||
</li> | </li> | ||
Revision as of 17:36, 3 October 2013
Calendar
Logic Gates Characterization
July 2013
- Week 4 (1-7 July)
04/07
Summary of all the work we have to do for the logic gates. These are the necessary genes we needed to cloned into the logic gates.
Fro And1: rbs-luxI and rbs-Ci-ter
For Xor1: rbs FimE ter
For And2: reversed rbs luxI
For Xor2: reversed rbs-RFP-ter
- Week 6 (15-21 July)
Amplification of fimE recombinase (rbs+fimE) from the Michigan Team.
Minipreps, and electrophoresis checking-> good amplification.
- Week 7 (22-28 July)
Problem with AND1, the synthesis is not correct. We cannot go on with this gate. We planned order a new synthesis.
Amplification of a new rbs-luxI (BBa_K516011). We realized that the first rbs –luxI we amplified was not the good one (wrong size).
Cloning of rbs+fimE with a terminator, wrong electrophoresis profile.
Cloning of rbs-luxI with rbs-CI-ter. This cloning step was successful.
Transfer of xor2 synthesis from puc54 to pSB1C3. We did not get xor2 inside pSB1C3, but only the backbone vector.
Insertion of reversed RFP inside AND2 (with cla1/bamH1). Electrophoresis profiles were not correct, so the insertion was not successful. It was probably a problem of restriction time.
August 2013
- Week 8 (29-04 August)
Cloning of rbs+fimE with a terminator. This time we succeeded.
Amplification of the fragment rbs luxI-rbs-CI-ter for the AND1 gate.
Insertion of rbs-FImE-ter inside the Xor1 gate. We made restriction controls with different enzymes (stu1,hindIII), but the results were not fruitful.
We tried again to introduce RFP inside AND2, still not working.
We succeeded to transfer Xor2 inside pSB1C3.
- Week 9 (05-11 August)
Still lot of problems to insert reversed RFP inside AND2. The transformation does not work. We needed to find a solution (strataclone? Infusion?...).
Insertion of reversed rbs luxi inside AND2. Same problem as for AND2-RFP.
Cloning of rbs-fimE-ter into Xor1. It did not work because of a ligation problem.
Insertion of reversed RFP into XOR2, no insertion, only the backbone vector was present.
- Week 10 (12-18 August)
As we faced lot of issues with the reversed RFP and reversed rbs-luxI, we decided to order new oligonucleotide in order to create new reversed fragments as we did during the week 6 PCR to invert RFP and rbs.LuxI with the restriction site ClaI BamHI, and purification.
We tested a wide range of elongation temperatures (60°C-75°C). We got the right fragments for the two biobricks with good size. But on the electrophoresis revelations we got one parasite fragment around 5kb.
- Week 11 (19-25 August)
We amplified the reversed fragment RFP and rbs luxI with the new oligos. We did not find the solution to avoid the parasite fragment so we used PCR clean-up & gel purification.
Insertion of these fragments into the gate (rbs-luxI into AND2 and RFP into XOR2 and AND2).
We did not get better results even with these new fragments.
We realized in fact we have a problem with the XOR2 gate. Instead of having only one site cla1 inside the XOR2, we had two. The problem was therefore the restriction with cla1!
Extraction And 1 gate from And gate part of Bonnet construction by PCR.
Cloning : (Digestion, Ligation, Transformation , Pre-culture, Miniprep )
Insertion of And 1 gate extracted by PCR into the plasmid backbone pSB1K3
Assembly of And 1 gate extracted by PCR and rbs RFP term part
Assembly of Xor 1 gate and rbs RFP term parts
September 2013
- Week 1 (02-08 Sept)
- Week 2 (09-15 Sept)
06/09
Cloning AND2 and XOR2 with RFPinv
08/09
Verfication of the cloning, AND2.RFPinv ok
10/09
Cloning AND2.RFPinv with PolT7 and XOR2 with RFPinv
11/09
In fusion to have XOR2 with RFPinv because cloning is not working
13/09
XOR2.RFPinv with in fusion OK
14/09
Cloning XOR2.RFPinv and AND2.RFPinv with the good PolT7 (with promoter)
15/09
XOR2.RFPinv and AND2.RFPinv with PolT7 ok for characterisation
