Team:KIT-Kyoto/Notebook-week6

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September 11th

The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

pSB1C3-UAS-1, -2, -3, -4	5uL
3buffer(NEB)	2uL
BglⅡ	0.5uL
dH₂O	12.5uL
Total	20uL

GAL4	40uL
4 buffer(NEB)	5uL
XbaⅠ(NEB)	0.5uL
SpeⅠ(NEB)	0.5uL
CIP	0.5uL
100×BSA	0.5uL
dH₂O	3uL
Total	40uL

	1uL
	2.5uL
	2.5uL
Ligation high	6uL
Total	12uL

	2uL
	2.5uL
	1.5uL
Ligation high	6uL
Total	12uL

September 12th

1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11).

1-1-47 Digestion of pSB1C3-UAS DNA by Spe1
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.

The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

Composition

pSB1C3-UAS-1, -2	40uL
3buffer(NEB)	5uL
SpeⅠ	1uL
100×BSA	0.5uL
dH₂O	3.5uL
Total	50uL

The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.

Photo of Agarose gel.

1-1-48 and 2-2-6 Ligation
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.

Ligation reactions are listed below.

pSB1C3-UAS (double digested with BglII and SpeⅠ)	1uL
EGFP fragments or LacZ fragments	2uL
EGFP or LacZ	2uL
Ligation high	2.5uL
dH₂O	2uL
Total	7.5uL

We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.

Composition

pSB1C3 (PCR) (double digested with XbaⅠand SpeI)	2uL
GAL4(double digested with BglⅡ and SpeⅠ)	2uL
HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ)	2uL
Ligation high	6uL
Total	12uL

1-1-49 and 2-2-7 Transformation of E. coli by Ligation products
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.

September 13th

The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

1-1-50 and 2-2-8 Isolating a single colony of E. coli carrying the candidate pSV1C3-UAS-LacZ or pSB1C3-HS-GAL4
We isolated colonies (one for pSB1C3-UAS-LacZ ,three for pSB1C3-HS-G4L4) and cultured in liquid medium(2.5ml LB Chloramphenicol(+)) at 37℃ for 16 hours. No transformed colony was detected for E. coli carrying the candidate pSB1C3-Act5C-GAL4.

1-2-8 Cut check for the candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP
The candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP DNAs (made 9/12) were digested with EcoRⅠ/ SpeⅠ and BglⅡ, respectively.

EcoRⅠand SpeⅠ

DNA sample	1uL
4 buffer(NEB)	0.5uL
EcoRⅠ-HF(NEB)	0.2uL
SpeⅠ(NEB)	0.2uL
100×BSA	0.05uL
dH₂O	3.25uL
Total	5uL

BglⅡ

DNA sample	1uL
3 buffer(NEB)	0.5uL
BglⅡ	0.2uL
dH₂O	3.3uL
Total	5uL

Then the digested samples were applied to Agarose gel electrophoresis in the order of no cut sample, EcoRⅠ/SpeⅠ-digested sample and BglⅡ-digested sample.

Result of electrophoresis

Results: No insert DNA was detected. Therefore we were not successful for these cloning.

1-1-51, 2-1-37 and 2-2-9. Ligation
Ligation of DNA fragments carrying GAL4 to pSB1C3 DNA was carried out for 2 hours at 16℃ in the reaction described below.

Composition

pSB1C3 (double digested with BglⅡand SpeⅠ)	1uL
GAL4 (double digested with BglⅡand SpeⅠ)	1.5uL
dH₂O	2.5uL
Ligation high	2.5uL
Total	7.5uL

Ligation of DNA fragments carrying G4L4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to SB1C3 DNA was carried out for 1 hours at 16℃ in the reaction described below.

Composition

pSB1C3(PCR)(double digested with XbaⅠand SpeⅠ)	2.5uL
GAL4(double digested with BglⅡand SpeⅠ)	2.5uL
HS or Act5c fragments (double digested with XbaⅠand BamHⅠ)	2uL
Ligation high	7uL
Total	14uL

Ligation of DNA fragments carrying EGFP or LacZ to pSB1C3-UAS was carried out for 1 hours at 16℃ in the reaction described below.

pSB1C3-UAS (double digested with BglⅡand SpeⅠ)	1uL
EGFP fragments or LacZ fragments (double digested with BglⅡand SpeⅠ)	2uL
dH₂O	2uL
Ligation high	5uL
Total	10uL

1-1-52, 2-1-38 and 2-2-10 Transformation of E. coli by Ligation products
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and cultured at 37℃ for 16 hours.

September 14th

The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

1-1-53 and 2-1-39 Isolating a single colony of E. coli transformed with Ligation products
We picked up three colonies of E. coli carrying the candidate pSB1C3-G4L4, two from the candidate pSB1C3-UAS-EGFP and three from the candidate pSB1C3-UAS-LacZ (made on 9/13), and cultured in 2.5mL LB Chloramphenicol(+) liquid medium at 37℃ for 16 hours.

1-1-54 and 2-2-11 Purification of the candidate pSB1C3-UAS-LacZ and pSB1C3-HS-GAL4 DNA
The pSB1C3-UAS-LacZ DNA and pSB1C3-HS-G4L4 DNA was purified from E. coli (cultivated on 9/12) by QIA prep Spin Miniprep Kit.

1-1-55 and 2-2-12 Cut check of the candidate pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, and pSB1C3-HS-GAL4 DNA
These candidate pSB1C3-UAS-EGFP DNA (prepared on 9/12), pSB1C3-UAS-LacZ DNA (prepared on 9/12), pSB1C3-UAS-LacZ DNA (prepared on 9/14) and pSB1C3-HS-G4L4 DNA (prepared on 9/14)

DNA sample	1uL
4 buffer(NEB)	1uL
EcoRⅠ-HF(NEB)	0.2uL
SpeⅠ(NEB)	0.2uL
100×BSA	0.1uL
dH₂O	7.5uL
Total	10uL

Digested samples were applied to the Agarose gel electrophoresis.
From the left side,
Two pSB1C3-UAS-EGFP DNA isolated from independent colonies (9/12) uncut , digested two pSB1C3-UAS-EGFP DNA (9/12),
pSB1C3-UAS-LacZ DNA (9/12) uncut, digested pSB1C3-UAS-LacZ DNA (9/12)cut,
pSB1C3-UAS-LacZ DNA (9/14) uncut, digested pSB1C3-UAS-LacZ DNA (9/14),
pSB1C3-HS-G4L4 DNA from three independent colonies (9/14) uncut, digested pSB1C3-HS-G4L4 DNA from three independent colonies (9/14)

Results

We found that pSB1C3-UAS-LacZ DNA (9/14) was successfully constructed !. This is the first one we successfully constructed as a Biobrick part.

2-2-13 Ligation
Ligation of DNA fragments carrying G4L4 and those carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA was carried out for 2 hours at 16℃ in a reaction described below.

pSB1C3(PCR) (double digested with XbaⅠand SpeⅠ)	2uL
GAL4 (double digested with BglⅡand SpeⅠ)	3uL
HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ)	1uL(HS) or 2.5uL(Act5c)
Ligation high	6uL(HS) or 7.5uL(Act5c)
Total	12uL(HS) or 15uL(Act5c)

2-2-14. Transformation of Ligation products
Ligation products were transformed into E. coli XL-1 blue.

September 15th

The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

1-1-56. Purification of candidate plasmid DNAs
We reproduced The candidate pSB1C3-UAS-LacZ DNA was purified from the three independent colonies, pSB1C3-UAS-EGFP DNA from two independent colonies, pSB1C3-G4L4 DNA from three independent colonies by QIA prep Spin Miniprep Kit.

Cut check of the candidate pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA
The candidate pSB1C3-UAS-LacZ DNA from two independent colonies, the candidate pSB1C3-UAS-EGFP DNA from two independent colonies and pSB1C3-G4L4 DNA from three independent colonies were double digested with EcoRI and SpeI.

Composition

DNA sample	1uL
4 buffer(NEB)	1uL
EcoRⅠ-HF(NEB)	0.2uL
SpeⅠ(NEB)	0.2uL
100×BSA	0.1uL
dH₂O	7.5uL
Total	10uL

The digested samples were applied to Agarose gel electrophoresis. From the left side lane to the right, two digested candidate pSB1C3-UAS-LacZ DNA, two digested candidate pSB1C3-UAS-EGFP DNA, three digested candidate pSB1C3-G4L4 are shown.

Results

We identified the properly constructed pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA on the gel ! These are Biobrick parts we successfully constructed.

September 16th

The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. In total 83 flies from microinjected embryos were mated with yw flies. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.

1-1-58 Preparation of Biobrick part DNAs to submit
E. coli carrying the successfully constructed pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA were cultured for 16 hours at 37℃.

September 17th

The progeny flies were inspected by dissecting microscope to look for the successfully transformed w+ red eye flies (red eye screening). However, no red eye fly was found.

1-1-59 Cut check of the Biobrick part DNAs
The pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA were purified by QIA prep Spin Miniprep Kit. These DNAs were double digested with EcoRI and SpeI again and the digested samples were applied to Agarose gel electrophoresis.

Results: The correct size of inserts were detected in the double digested samples further confirming that the pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA are properly constructed.

1-1-60 Submission of the Biobrick parts
The plasmids pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, pSB1C3-G4L4 DNA and the previously prepared pSB1C3-UAS were sent out to the iGEM Headquarter via FedEx.

1-1-61 Preparation of DNA for transfection into cultured Drosophila cells
E. coli carrying pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP were grown for 16 hours at 37℃.

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