From 2013.igem.org

Calendar

Logic Gates

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
Week 12 (26-31 August)

Creation of another reversed RFP fragment but with bamH1 restriction site on each side of this DNA fragment. With this, we were able to overcome the problem of two cla1 restriction site inside XOR2. We cloned this fragment into XOR2. But it didn’t work.
It seemed that RFP was well inserted into AND2, but after DNA sequencing analysis, we didn’t get the right fragment at the right place.
First verification of cloning parts size by digestion by EcoRI and PstI enzymes:

o And 1 – pSB1K3
- pSB1K3 linearized: about 2200 bp.
- And 1 fragments: about 500 bp.
o Xor 1 - rbs RFP term – pSB1K3
- pSB1K3 linearized: about 2200 bp.
- Xor 1 - rbs RFP term fragments: about 1200 bp.
o And 1 - rbs RFP term – pSB1C3
- pSB1C3 linearized: about 2000 bp.
- And 1 - rbs RFP term fragments: about 1200 bp.

=> Cloning parts size is confirmed.

• Second verification of cloning parts by digestion:
o Xor 1 - rbs RFP term digested by EcoRI and BstXI => 2 fragments: about 1007 bp and 2370 bp .
o And 1 – rbs RFP term digested by EcoRI and BstXI => 2 fragments: about 1012 bp and 2350 bp.
o And 1 – pSB1K3 digested by EcoRI and PsiI => 2 fragments: about 266 bp and 2440 bp.

=> Cloning parts are confirmed.
• Third verification of cloning parts by sequencing
=> And 1 – pSB1K3, Xor 1 - rbs RFP term, And 1 – rbs RFP term constructions are confirmed