From 2013.igem.org

Calendar

Blue Light Sensor Characterization

June 2013

Week 3 (24-30 June)
Amplification of new biobricks for cloning:
BBa_ J23116 promoter,
TetR,
pTet promoter,
YF1-FixJ,
BBa_B1010 terminator,
pFixK2 promoter
mRFP1.

July 2013

Week 4 (1-7 July)
At the beginning, the following construction was planned to describe the behavior of the couple YF1-FixJ/pFixK2 under two conditions: light and dark. YF1-FixJ was designed under the control of tetR and pTet, the general inducer system.

A first round of assembling was started up thanks to the iGEM 3A Assembly method:
- (1) promoter (Bba_J23116) + tetR (BBa_P0440) in PSB1K3 (875 bp)
- (2) YF1-FixJ (BBa_K592016) + terminator (BBa_B1010) in PSB1A2 (1836 bp)
- (3) pFixK2 (BBa_K592006) + RFP (Bba_k081014) in PSB1A2 (1024 bp)

Transformation into DH5α competent cells and miniprep of the clones obtained. The electrophoresis of the digested plasmids with EcoRI and PstI shows the presence of consistent inserts size for (1) promoter (Bba_J23116) + tetR (BBa_P0440) and (2) YF1-FixJ (BBa_K592016) + terminator (BBa_B1010). Presence of undigested plasmids was noted (a third band corresponding to the couple vector-insert was pointed out). No consistent result was seen for (3) pFixK2 (BBa_K592006) + RFP (Bba_k081014) (lack of 250 bp corresponding to pFixK2 size)
Week 5 (8-14 July)
With the previous results, a second round of cloning was launched with the 3A Assembly method:
- (3) pFixK2 (BBa_K592006) + RFP (Bba_k081014) in PSB1K3 instead of PSB1A2 (1024 bp)
- (4) promoter + tetR (Bba_J23116 + BBa_P0440) + ptetR (BBa_R0040) in PSB1C3 (929 bp)
The restriction of the minipreps with EcoRI and PstI confirmed the presence of correct inserts size for the two constructions.
Week 6 (15-21 July)
Thanks to the previous assembled biobricks, a third round of cloning was done:
- (5) promoter + tetR + ptetR (Bba_J23116 + BBa_P0440 + BBa_R0040) + YF1-FixJ + terminator (BBa_K592016 + BBa_B1010) in PSB1K3 (2725 bp)

The clones were minipreped. The gel electrophoresis of the digested purified plasmids with EcoRI and PstI doesn’t show correct size inserts.
In parallel, plasmid backbones were amplified.
Week 7 (22-28 July)
New attempts to assemble construction (5): promoter + tetR + ptetR with YF1-FixJ + terminator but cloning failed because of an improper restriction of construction (4) (promoter + TetR + pTet) by EcoRI and SpeI.

These series of failed cloning make us think about solutions to improve efficiency of our assemblies.
- Unphosphorylate plasmid backbones to avoid ligation-type vector-vector:

- Change antibiotic resistances (often, part A and part B used for assembly have the same antibiotic resistance)
- Test out a bipartite assembly method instead of the common 3A assembly method:
o Option 1: cutting part A with SpeI/PstI and part B with XbaI/PstI
o Option 2: cutting part A with EcoRI/SpeI and part B with EcoRI/XbaI.

August 2013

Week 8 (29-4 August)
Assemblies with the bipartite method can start!
A new round of construction (5) was launched but the gel electrophoresis of the minipreps cut by EcoRI and PstI doesn’t show any correct inserts (again!).

Indeed, several restrictions with different couples of enzymes was done to check the prefix and the suffix in construction (4): this control shew that the SpeI restriction site didn’t work.

Thus, we choose to assemble construct (2): YF1-FixJ+term with construct (3): pFixJ-RFP (construct (6)) and send to sequencing previous constructs obtained. Construct 6 didn’t work.
Week 9 (5-11 August)
Sequencing analysis of previous constructs:

- Construction (4): presence of the prefix, the suffix, the promoter J23116 and pTet but TetR in the wrong orientation.
- Construction (2):presence of the prefix, the suffix, YF1-FixJ but lack of the terminator B1010
- Construction (3): presence of the prefix, the suffix, pFixK2 and RFP

Only construction (3) worked as expected. New strategy was done to obtain a construction to characterize the blue light sensor in time and a new construction without the general inducer system was chosen:
Week 10 (12-18 August)
New assemblies were launched:
- (7): Promoter J23116 with YF1-FixJ
- (8): terminator B1010 with construction (3)

Gel electrophoresis shew corrects sizes for part digested by EcoRI and PstI. But both constructions were checked in second time in order to make sure that short parts were correctly assemblied (3.5% of agarose)
- Construction (7) cut by EcoRI and PvuII should give an insert of 111 bp ( 76 bp if it is without the promoter)
- Construction (8) cut by EcoRI and HpaI shoud give an insert of 415 bp (367 bp if it is without the terminator: the part pFixK2-RFP sequenced and used for the cloning was also cut by this couple of enzyme to make a reference)
Constructions (7) and (8) were verified.
Week 11 (19-25 August)
The last cloning was done: assembling of construction (7) and construction (8). And after a lot of clonings, minipreps, and agarose gels, we had the final construction to characterize the blue light sensor:
Week 12 (26-31 August)
Attempts to characterize the blue light sensor in darkness and in light conditions.
With this construction, colonies were supposed to express the modified RFP in darkness and to inhibit this expression in light. Several subcultures were done to ensure that colonies were in light/dark states for at least 3 subcultures.
Visually, colonies in darkness didn’t show any difference with colonies in light. Measures with a fluorometer shew the same results. We supposed that the promoter wasn’t strong enough to express YF1-FixJ in large quantity, thus our biological construction didn’t works expected.

September 2013

Week 13 (1-8 September) & Week 14 (9-15 September)
Several cloning were done to put a strong promoter (Bba_J23119) instead of the too weak promoter (Bba_J23116).
Cloning failed for the final construction with the strong promoter but we manage to have construction (9): YF1-FixJ-terminator-pFixK2-RFP (digested miniprep with EcoRI and PstI shew corrects sizes for 7 clones)