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• YcgF Gene, naturally available in [http://ecoliwiki.net/colipedia/index.php/ycgF:Gene 3 E. Coli strains]. Can use the promotor of Ycgf gene for expression
• Probable strain K12
• Promotor voor YcgF gene:
  • AACAATCCAGGGTAATGGGTGAGGCGAGAGTAAGACGGTAACAGACATATCTTCTTG TGTCTTTCTTTTAATACCAAAACATAACCGTTTCTTTACATTGATAAAAAATGGAAAAAG TTGAACACTAGTTGGCGAAAAATCTTGTATAGATTGTCAGTTAAATGATGCAATATGTT TTATCATAACACATTGTTTTATATGCATTAGCACTAATTGCAAAAAATTAATTTATCATT CTGTACACATATTTCGTACAAGTTTGCTATTGTTACTTCACTTAACATTGATTAACATTTTTAACAGAGGCGTAGCATG
    (source: [http://www.ncbi.nlm.nih.gov])

• Primer selection for the promotor [Done]
  • Send mail to Institut for Biologie-Mikrobiologie ([http://www.ncbi.nlm.nih.gov/pubmed/19240136 The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli]) [Done]

• Mail Institut fur Mikrobiologie der Westfalischen, Wilhelms-Universitat Munster for the plasmid [Done]

• Primers for the blue light receptor have arrived

• PCR on promoter region in MC4100 E.coli colony using primers and tested on agarose gel

• concentration: 91,9 ng/μl
• 260/280 = 1,90

rbs + GFP + terminator was plated.

was cultured in liquid medium and put in the 37°C incubator overnight

GFP ()

• Miniprepped and nanodropped
  • Concentration: 85,6ng/μl
  • 260/280: 1,87
• A restriction digest was performed to cut the plasmid with EcoRI and XbaI
  • A mixture of 20μl was made(3x):6μl DNA, 2μl bufferH, 1μl EcoRI and 1μl XbaI, 10μl AD
  • The mixture was incubated for at least an hour at 37°C

BLR promoter region The PCR product that was purified friday (24/07) is digested with EcoRI and partially digested with SpeI

• Digestion with EcoRI
  • Following mixture was made (x6): 5μl DNA, 2μl buffer H, 1μl EcoRI, 12μl MilliQ
  • Incubated for 1h at 37°C
• Partial digestion with SpeI
  • Dilution of the enzymes:
    • AD/b: 225μl MQ + 25μl buffer H
    • 1/100: 1μl SpeI + 99μl AD/b
    • 1/200: 50μl 1/100 + 50μl AD/b
    • 1/500: 20μl 1/100 + 80μl AD/b
  • Made following mixture:

• The mixture was incubated for 15 min at 37°C

After the restriction digests, the products had to be checked for their length. So, an agarose gel was run for both the cut GFP-plasmids and the cut promoter region. A photograph was taken and following conclusions were made:

• Plasmids with GFP appeared to have cut decently
• Promoter region: the partial digestion did not seem to have done the trick. We only found a lane around 360 bp while we expected to find another lane just under 200 bp due to cutting at the "forbidden" SpeI site in the middle. As we did not find this lane in our gel we concluded that SpeI probably did not cut. This might be because it was diluted too much or because we did not incubate long enough.

We concluded to purify both the plasmids and the promoter region through gel extraction. After nanodropping, we had these results:

• Plasmids:
  • Concentration: 22ng/µl
  • 260/280: 1,83
• Promoter region (two samples):
  • Concentration: 31,7ng/µl and 32,7ng/µl
  • 260/280: 1,84 and 2,06

The conclusion of the day was to redo a partial digestion on the promoter regions, this time under different conditions (longer incubation time and less diluted). At the same time a Knelow technique would be used on the newly PCR-ed promoter region in order to cut out the SpeI site.

Later that evening we received an email from Regine Hengge (co-author on the article [http://www.ncbi.nlm.nih.gov/pubmed/19240136 The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli]). This contained valuable information about the location of the actual promoter in our purified region.

• Concentration:
  • 192,7 ng/µl
  • 161,9 ng/µl
  • 155,1 ng/µl
• 260/280
  • 1,82
  • 1,82
  • 1,80

• concentration of clean pcr product.:
  • 102,6 ng/µl
  • 1,88

1. Restriction digest
   • cut with EcoRI and SpeI, incubation for 1,5 hour
2. Gel electrophoresis of
   • The cut piece should be 109 bp
3. Restriction digest
4. Electroporation of parts - and in electrocompetent cells

1. Gel electrophoresis of BlP cut with EcoRI and SpeI
2. Gel extraction of the Blp

Nanodrop results

1. PCR (with Pfx) of blue light promoter with primers iGEM 2260 and iGEM 2261
   • Annealing of 58C
2. Inoculate liquid medium
   • Inoculation of - and
3. Ligation

1. PCR product of 12-august of pairt
2. Miniprep of

Nanodrop results:

1. Restriction digest cut with EcoRI and XbaI
2. Electroporation of + and the ligation of ...
3. Gelelectrophoresis of
   • Only 1 band, so probably everything was cut
4. Gelextraction of
   • Nanodrop = 29,3 ng/μl
5. Ligation of vector and insert

1. Transfer of the plates with cells from the ligation: 20 colonies have been transferred to a new plate to achieve more single colonies
2. Plating of from -80°C
3. Electroporation of the blue light promotor ligation (with ) in new competent cells

Lig B has been re-ented on a new plate because too many cells had grown on the one we made on aug 14. We always used single kolonies to re-ent.

2) Restriction digest of the DNA from step 1 with EcoRI and PstI

3) Gel electrophoresis to check if the insert in the plasmid is the correct size, which it was, so the ligation was succesful.

1. Plates with LigA were put under blue light. The LEDs were put on their max capacity.
2. Restriction digest with
   • tubes (1,3,5,7,9) of LigA (BLP + ) cut with EcoRI en PstI
   • promotor cut with EcoRI en SpeI (4x)
3. Gel electrophoresis with the RD of LigA and followed by a gel extraction:
   • Note: tube 5 of LigA was loaded poorly on the gel and could not be used.
   • Note: the samples of promotor were barely visible. Only 2 of the 4 samples were recovered by extraction.

4. Enting of liquid cultures with kanamycin and

• Miniprepping the liquid cultures with

• Restriction digest of with EcoRI and PstI
• Gel electrophoresis and extraction of the RD of

--> failed. There was no signal at all

• New colonies were ented in liquid culture.
• Ligation C performed: (A) +

1. The plates with ligation A (blp + GFP) were fetched from the blue light installation. There was no GFP signal. The following actions will be taken:
   • The plasmids will be purified from the colonies and will be sequenced using primer 2260.
   • Next time, we will probably put them in liquid cultures under the LED's while shaking gently. Also, other parameters that need to be considered are being researched.
   • They will not be exposed to the light as long anymore. We decided that 1h will be more than enough.
   • Possible bleaching?
2. A colony from all three plates with the LigA-construct was taken and ented in liquid culture. This was needed to check whether the colonies were still alive.
   • culture 13/08
   • culture 14/08 (1)
   • culture 14/08 (2)
3. By 5pm, one of the liquid cultures had grown and was miniprepped. 20μl was sent for sequencing together with 5μM of primer 2260.

4. Two electroporations were performed and plated on LB medium. One with LigC ( + ) and one with DNA.

1. The electroporations of 26/08 were checked.
   • had some colonies. they were ented in liquid culture.
   • LigC ( + ) did not grow. we figured that an insert of 35bp was too short to ligate so we decided to use as insert and + as vector . The following restriction digest was started:
     • was cut with SpeI and PstI
     • was cut with XbaI and PstI
2. The miniprep that was made to sequence the LigA construct on 26/08 was used again to perform a restriction digest (EcoRI and PstI). This was put on gel to check whether there actually was LigA-insert in the vector and whether the insert had the right length. The gel showed a signal at 1000 bp and at 2000 bp which coincides with the insert (BLp + GFP) and the vector.
3. A new setup to light the E.coli was engineered.
   • Fresh cultures were made from the old ones (LB plate ligA 14/08 and the two liquid cultures from 26/08 were used as templates.)
   • They were put in the 16°C room for about an hour
   • A blue light (40W) was put on them for about an hour
   • They were put in the 37°C incubator overnight

1. Miniprep of liquid cultures of grown overnight.

1. Gel electrophoresis of miniprep and of restriction digest from 27/08 ( and . There was an unexpected signal at 800bp at the ( lanes. The conclusion was that this part can not be used and we have to start all over from the basic DNA to make this ligation C.
2. We checked our cultures that were radiated with blue light on 27/08.
   • The liquid cultures were put in the FACs machine. There was some fluorescence which is probably due to GFP. However, it can not be excluded that this is due to leakage from our promotor. So, a new set up needs to be made where decent controls are included.
   • The LB plates were put under a special GFP lamp. Just like the liquid cultures, there was signal but the same considerations need to be made.
3. A new plan was designed in order to make a permanent glycerol stock of LigA and to make a new set up for GFP measurements, this time including controls.
   • LigA and were electroporated, plated and put overnight in the 37°C incubator.
   • was also plated starting from a glycerol stock and grown overnight at 37°.

The following was done over the weekend:

• Saturday:
  • LigA grew well overnight. 4 colonies were selected for further testing and plated on new plates
  • The plates with overgrew, so a single colony was picked and replated
  • The plates containing electroporated ligC from 26/08 had two colonies after all. These were ented in liquid cultures and grown overnight, so it can be checked later if these colonies do in fact contain ligC.
• Sunday:
  • The 4 colonies that were selected on Saturday grew well and were ented in liquid cultures.
  • Some colonies from were also ented in liquid culture.

1. A miniprep and RD (with EcoRI and PstI) were performed on the four colonies that were ented on Sunday.
   • Nanoprop results
   • RD results on gel: the right signals were detected

1. LigA was plated on LB medium. This will be the "motherplate" from which the glycerol stock and the testing cultures will be taken from.
2. Electroporation of in competent cells. It was plated and put overnight in the incubator.
3. In order to be able to grow vector , which contains the toxic ccdB gene, we need special cells which carry the gyrA462 mutation. This is strain DB3.1 from E.coli. These were plated and put in the incubator overnight.

1. Experimental set up for irradiation of LigA ( + )
   • Enting colonies from the motherplate:
     • 4 agar plates with LigA = 2 for irradiation and 2 for control.
     • 4 liquid cultures with LigA = 2 for irradiation and 2 for control.
     • 4 agar plates with = 2 for irradiation and 2 for control.
     • 4 liquid cultures with = 2 for irradiation and 2 for control.
   • There are two groups, each with 8 cultures.
     • Shift one: 2 agar plates and 2 liquid cultures with LigA and 2 agar plates and 2 liquid cultures with were put in 16°C for one hour. After an hour, half of them were irradiated with blue light for an hour while the other have was wrapped in aluminium foil as a control. Afterwards they are put in the 37°C incubator for some time
     • Shift two: they were put in the 37°C for some hours so that the cultures can grow.
   • After measurement of the first shift the following was concluded:
     • Although the cells with had a small signal after FACS, the plates showed no GFP when put under the lamp.
     • LigA that was not exposed to blue light had the same amount of signal as the construct that had been exposed. we assume that this is due to the exposure to white light when the cultures are exposed. Possibly, the promotor was then already activated since white light contains blue light frequencies. A new set up will be made over the weekend where all cultures will be made in a dark room.
     • Dilutions of the shift one liquid cultures were made + 2 new cultures from the motherplate from ligA. These were put overnight under blue light together with the cultures of shift 1
2. Following was ented in liquid culture:
   • LigA from motherplate
   • from motherplate
   • DB3.1

1. FACS measurements:
   • The cultures from shift 2 had similar results as shift one. However, the GFP signal measured from the LigA construct was stronger. Thus, cells definitely need to grow before undergoing irradiation.
2. The dilutions and the shift one cultures put overnight did not show any significant results.
3. was miniprepped and nanodropped

1. After nanodropping, was restricted with SpeI and PstI, while was cut with PstI and XbaI.
2. A gel electrophoresis and extraction was performed. This showed an unexpected 800 bp signal at the lanes with . This is in fact due to the vector in which this part is ligated. contains a reporter gene, RFP. Thus, in combination with the promotor, a construct is formed that can be used to measure activity of this promotor. Since the promotor is flanked by standard assembly restriction sites we can replace the current promotor () by any other promotor, for instance .
3. DB3.1, and LigA were ented in liquid culture and grown overnight to make glycerol stocks and competent cells

1. A restriction digest on with EcoRI and SpeI was performed. This will cut the promotor out of its vector () which has a RFP reporter gene. We will put (cut with EcoRI and SpeI) in this vector. This way we will get a construct where RFP will be produced dependant on our blue light promotor. We can then compare this with the intensity of RFP produced by .
2. Glycerol stock was made from LigA and . They are stored in the -80°C in S&P F8 67-68 and 69-70.
3. Ecoli strain DB3.1 cells were made competent for electroporesis.
4. Gel electorphoresis and extraction on

1. Gel extraction performed on the signals from 3/09
   • Nanodrop: 14,7 ng/µl of with a 260/280 of approx 2,12
2. Ligation with and (ligX 1)
3. A new restriction digest on and with EcoRI and SpeI was performed.
4. Gel extraction of these new digests. was nanodropped with a concentration of 28 ng/µl. this was ligated with (ligX 2)
5. Electroporation of

1. Restriction digest with EcoRI and PstI on and the purified pcr product of in order to get our promotor on itself in the standard vector.
2. Gel electroforesis and extraction of this RD. The concentration of this extraction was too low so no ligation was performed with these samples.
3. Extra pcr was done on with primers 2260 and 2261.
4. Liquid cultures of LigX and of were made from the 4x plates.

1. Dilutions of the liquid cultures of ligX (7/09) were made and put in 37°C to grow during the day.
2. In the evening the liquid cultures were first put in 16°C and then put under light.
3. The miniprep of LigX was not performed correctly.
4. Liquid cultures of were made.

1. FACS-ing the liquid cultures that were put under blue light on 08/09 gave no significant results.
2. Motherplate for was made
3. liquid cultures for checking the ligX were made.
4. LC with were miniprepped, nanodropped, restriction digested and put on gel. The signals were good. The vector, pSB1A2, was cut from the gel and gel extracted (10ng/µl). This was ligated with (LigY).

Nanodrop:

1. Since the results from our light experiments keep failing, some new ideas were computed. pSB1A2 is the backbone for our construct but since this is a high copy number plasmid perhaps the ratio of promotor to repressor is unequal (not enough repressor). A new enting in LC was done with ligX. These were grown for a few hours at 37°C and then put in 16°C room overnight so that more repressor can be made (its promotor is temperature-sensitive).
2. Ligation from 09/08 was electroporated in competent cells.
3. Miniprep of LigX, restriction digest and gel electrophoresis. The signals were good so a motherplate for ligX was made. Also, the vector pSB1A2 was extracted from the gel (39,1 ng/µl).

Nanodrop results:

1. The electroporated ligY did not grow. A new restriction digest on pcr product was performed with EcoRI and PstI.This was put on gel. There was a good signal so it was used to perform a ligation with vector pSB1A2.
2. A liquid culture that was made from pSB3K3 a few days ago showed growth after all. This was miniprepped and restriction digested to check whether the vector is actually present.
3. The liquid cultures with our blue light construct (lig) that stood overnight at 16°C was FACS-ed. Half of the cultures were exposed to blue light during the day and put under the FACS in the evening.

1. miniprep of ligY colonies. nannodrop: 111 ng/µl and 116 ng/µl.
2. restriction digest of ligY miniprep
3. gelelectrophoresis of restricted ligY and compared to ligA. the ligY fragment was larger than ligA which means that the ligation Y fauled. the promotor region probably ligated to RFP which was not restricted out of the vector properly.
4. gel extraction of plasmid pSB1A2 (nanodrop: 10ng/µl)
5. a new ligY was performed
6. control of pcr-product . It was ok.

1. miniprep of pBR322 (nanodrop: 24,5ng/µl)
2. restriction digest of pBR322 with EcoRI and PstI
3. gelelectrophoresis and extraction of pBR322 (nanodrop: 10,8ng/µl)
4. ligation of pBR322 with ligA (blp + )
5. electroporation of cells with LigY (attempt 3)

1. miniprep of pRB322 (nanodrop: 59,8ng/µl)
2. ligY was replated
3. logA electroporation
4. plating of pSB3K3 from -80°C freezer.

1. electroporation of logA (ligA in pBR322, a low copy plasmid).
2. a PCR on pSB3K3 with primers ......
3. restriction digest of pSB3K3, pBR322 and ligA with EcoRI and PstI followed by a gel electrophoresis and extraction.
4. ligations of ligA (blp+GFP) with pBR322 (logA) and pSB3K3 (likA) were put overnight
5. was taken from the distribution plates and plated on LB
6. liquid cultures of ligY (blp) were made in the morning, grew and were miniprepped in the evening followed by a restriction digest with EcoRI and PstI

1. gel electrophoresis of the RD of ligY and ligA. all of the inserts were ok and ligA had a right signal.
2. a motherplate for ligY was made
3. likA and LogA ligations were electroporated
4. a new ligation of likA and logA were made and put overnight.