Team:KIT-Kyoto/Notebook-week6

From 2012.igem.org

(Difference between revisions)
Line 104: Line 104:
-
<strong>*Parts</strong>
 
-
<br><br>
 
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　pSB1C3-UAS-1, -2, -3, -4　</Td><Td>　5uL　</Td></Tr>
<Tr><Td>　pSB1C3-UAS-1, -2, -3, -4　</Td><Td>　5uL　</Td></Tr>
Line 154: Line 152:
<h2>September 12th</h2>
<h2>September 12th</h2>
<br>
<br>
 +
<strong>1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs</strong>
 +
<br>
 +
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).
 +
<br><br>
 +
<strong>1-1-47 Digestion of pSB1C3-UAS DNA by Spe1</strong>
 +
<br>
 +
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.
 +
<br><br>
 +
<strong>*TNFA and API2</strong>
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
Line 160: Line 167:
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
Composition
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　pSB1C3-UAS-1, -2　</Td><Td>　40uL　</Td></Tr>
<Tr><Td>　pSB1C3-UAS-1, -2　</Td><Td>　40uL　</Td></Tr>
Line 171: Line 179:
</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.
 +
<br><br>
 +
Photo of Agarose gel.
 +
<br>
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/a/ab/0912kit.png" width="500" height="300">
<br><br>
<br><br>
-
 
+
<strong>1-1-48 and 2-2-6 Ligation</strong>
 +
<br>
 +
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.
 +
<br><br>
 +
Ligation reactions are listed below.
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　1uL　</Td></Tr>
+
<Tr><Td>　pSB1C3-UAS (double digested with BglII and SpeⅠ)　</Td><Td>　1uL　</Td></Tr>
-
<tr><td></td><Td>　2uL　</Td></tr>
+
<tr><td>　EGFP fragments or LacZ fragments　</td><Td>　2uL　</Td></tr>
-
<Tr><Td>　EGFP or LacZ　</Td><Td>　2uL　</Td></Tr>
+
<Tr><Td>　EGFP or LacZ　</Td><Td>　2uL　</Td></Tr><tr>
<td>　Ligation high　</td><Td>　2.5uL　</Td></tr><tr>
<td>　Ligation high　</td><Td>　2.5uL　</Td></tr><tr>
 +
<td>　dH<sub>2</sub>O　</td><Td>　2uL　</Td></tr><tr>
<td>　Total　</td><Td>　7.5uL　</Td></tr>
<td>　Total　</td><Td>　7.5uL　</Td></tr>
</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.
 +
<br><br>
 +
Composition
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　2uL　</Td></Tr>
+
<Tr><Td>　pSB1C3 (PCR) (double digested with XbaⅠand SpeI)　</Td><Td>　2uL　</Td></Tr>
-
<tr><td></td><Td>　2uL　</Td></tr>
+
<tr><td>　GAL4(double digested with BglⅡ and SpeⅠ)　</td><Td>　2uL　</Td></tr>
-
<Tr><Td></Td><Td>　2uL　</Td></Tr>
+
<Tr><Td>　HS fragments or Act5c fragments  (double digested with XbaⅠand BamHⅠ)　</Td><Td>　2uL　</Td></Tr><tr>
<td>　Ligation high　</td><Td>　6uL　</Td></tr><tr>
<td>　Ligation high　</td><Td>　6uL　</Td></tr><tr>
<td>　Total　</td><Td>　12uL　</Td></tr>
<td>　Total　</td><Td>　12uL　</Td></tr>
</Table>
</Table>
<br><BR>
<br><BR>
 +
<strong>1-1-49 and 2-2-7  Transformation of E. coli by Ligation products</strong>
 +
<br>
 +
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℃.
 +
<br><br>
<h2>September 13th</h2>
<h2>September 13th</h2>
Line 200: Line 224:
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
<br><br>
<br><br>
-
 
+
<strong>1-1-50 and 2-2-8 Isolating a single colony of E. coli carrying the candidate pSV1C3-UAS-LacZ or pSB1C3-HS-GAL4</strong>
-
 
+
<br>
-
<strong>*Parts</strong>
+
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.
<br><br>
<br><br>
 +
<strong>1-2-8 Cut check for the candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP</strong>
 +
<br>
 +
The candidate pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP DNAs (made 9/12) were digested with EcoRⅠ/ SpeⅠ and BglⅡ, respectively.
 +
<br><br>
 +
EcoRⅠand SpeⅠ
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　DNA sample　</Td><Td>　1uL　</Td></Tr>
<Tr><Td>　DNA sample　</Td><Td>　1uL　</Td></Tr>
Line 214: Line 244:
</Table>
</Table>
<BR><BR>
<BR><BR>
-
 
+
BglⅡ
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　DNA sample　</Td><Td>　1uL　</Td></Tr>
<Tr><Td>　DNA sample　</Td><Td>　1uL　</Td></Tr>
Line 222: Line 253:
<td>　Total　</td><Td>　5uL　</Td></tr>
<td>　Total　</td><Td>　5uL　</Td></tr>
</Table>
</Table>
-
 
+
<br><br>
 +
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.
 +
<br><br>
 +
Result of electrophoresis
 +
<br>
<img src="https://static.igem.org/mediawiki/2012/3/3f/0913kit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/3/3f/0913kit.png" width="500" height="300">
<br><br>
<br><br>
-
 
+
Results: No insert DNA was detected. Therefore we were not successful for these cloning.
 +
<br><br>
 +
<strong>1-1-51, 2-1-37 and 2-2-9. Ligation</strong>
 +
<br>
 +
Ligation of DNA fragments carrying GAL4 to pSB1C3 DNA was carried out for 2 hours at 16℃ in the reaction described below.
 +
<br><br>
 +
Composition
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　1uL　</Td></Tr>
+
<Tr><Td>　pSB1C3 (double digested with BglⅡand SpeⅠ)　</Td><Td>　1uL　</Td></Tr>
-
<tr><td></td><Td>　1.5uL　</Td></tr>
+
<tr><td>　GAL4 (double digested with BglⅡand SpeⅠ)　</td><Td>　1.5uL　</Td></tr>
<Tr><Td>　dH<sub>2</sub>O　</Td><Td>　2.5uL　</Td></Tr><tr>
<Tr><Td>　dH<sub>2</sub>O　</Td><Td>　2.5uL　</Td></Tr><tr>
<td>　Ligation high　</td><Td>　2.5uL　</Td></tr><tr>
<td>　Ligation high　</td><Td>　2.5uL　</Td></tr><tr>
Line 234: Line 276:
</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
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.
 +
<br><br>
 +
Composition
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　2.5uL　</Td></Tr>
+
<Tr><Td>　pSB1C3(PCR)(double digested with XbaⅠand SpeⅠ)　</Td><Td>　2.5uL　</Td></Tr>
-
<tr><td></td><Td>　2.5uL　</Td></tr>
+
<tr><td>　GAL4(double digested with BglⅡand SpeⅠ)　</td><Td>　2.5uL　</Td></tr>
-
<tr><td></td><Td>　2uL　</Td></tr>
+
<tr><td>　HS or Act5c fragments (double digested with XbaⅠand BamHⅠ)　</td><Td>　2uL　</Td></tr>
<td>　Ligation high　</td><Td>　7uL　</Td></tr><tr>
<td>　Ligation high　</td><Td>　7uL　</Td></tr><tr>
<td>　Total　</td><Td>　14uL　</Td></tr>
<td>　Total　</td><Td>　14uL　</Td></tr>
</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
Ligation of DNA fragments carrying EGFP or LacZ to pSB1C3-UAS was carried out for 1 hours at 16℃ in the reaction described below.
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　1uL　</Td></Tr>
+
<Tr><Td>　pSB1C3-UAS (double digested with BglⅡand SpeⅠ)　</Td><Td>　1uL　</Td></Tr>
-
<tr><td></td><Td>　2uL　</Td></tr>
+
<tr><td>　EGFP fragments or LacZ fragments (double digested with BglⅡand SpeⅠ)　</td><Td>　2uL　</Td></tr>
<Tr><Td>　dH<sub>2</sub>O　</Td><Td>　2uL　</Td></Tr><tr>
<Tr><Td>　dH<sub>2</sub>O　</Td><Td>　2uL　</Td></Tr><tr>
<td>　Ligation high　</td><Td>　5uL　</Td></tr><tr>
<td>　Ligation high　</td><Td>　5uL　</Td></tr><tr>
Line 252: Line 298:
</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
<strong>1-1-52, 2-1-38 and 2-2-10 Transformation of E. coli by Ligation products</strong>
 +
<br>
 +
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.
 +
<br><br>
<h2>September 14th</h2>
<h2>September 14th</h2>
Line 261: Line 310:
<br><br>
<br><br>
-
 
+
<strong>1-1-53 and 2-1-39 Isolating a single colony of E. coli transformed with Ligation products</strong>
-
<strong>*Parts</strong>
+
<br>
 +
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.
 +
<br><br>
 +
<strong>1-1-54 and 2-2-11 Purification of the candidate pSB1C3-UAS-LacZ and pSB1C3-HS-GAL4 DNA</strong>
 +
<br>
 +
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.
 +
<br><br>
 +
<strong>1-1-55 and 2-2-12 Cut check of the candidate pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, and pSB1C3-HS-GAL4 DNA</strong>
 +
<br>
 +
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)
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
Line 273: Line 331:
<td>　Total　</td><Td>　10uL　</Td></tr>
<td>　Total　</td><Td>　10uL　</Td></tr>
</Table>
</Table>
 +
<br><br>
 +
Digested samples were applied to the Agarose gel electrophoresis.
 +
<br>
 +
From the left side,<br>
 +
Two pSB1C3-UAS-EGFP DNA isolated from independent colonies (9/12) uncut , digested two pSB1C3-UAS-EGFP DNA (9/12),
 +
<br>
 +
pSB1C3-UAS-LacZ DNA (9/12) uncut, digested pSB1C3-UAS-LacZ DNA (9/12)cut,
 +
<br>
 +
pSB1C3-UAS-LacZ DNA (9/14) uncut, digested pSB1C3-UAS-LacZ DNA (9/14),
 +
<BR>
 +
pSB1C3-HS-G4L4 DNA from three independent colonies (9/14) uncut, digested pSB1C3-HS-G4L4 DNA from three independent colonies (9/14)
 +
<br><br>
 +
Results
 +
<br>
<img src="https://static.igem.org/mediawiki/2012/e/ec/0914kit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/e/ec/0914kit.png" width="500" height="300">
<br><br>
<br><br>
-
 
+
We found that pSB1C3-UAS-LacZ DNA (9/14) was successfully constructed !. This is the first one we successfully constructed as a Biobrick part.
 +
<br><br>
 +
<strong>2-2-13 Ligation</strong>
 +
<br>
 +
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.
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　2uL　</Td></Tr>
+
<Tr><Td>　pSB1C3(PCR) (double digested with XbaⅠand SpeⅠ)　</Td><Td>　2uL　</Td></Tr>
-
<tr><td></td><Td>　3uL　</Td></tr>
+
<tr><td>　GAL4 (double digested with BglⅡand SpeⅠ)　</td><Td>　3uL　</Td></tr>
-
<tr><td></td><Td>　1uL(HS) or 2.5uL(Act5c)　</Td></tr><tr>
+
<tr><td>　HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ)　</td><Td>　1uL(HS) or 2.5uL(Act5c)　</Td></tr><tr>
<td>　Ligation high　</td><Td>　6uL(HS) or 7.5uL(Act5c)　</Td></tr><tr>
<td>　Ligation high　</td><Td>　6uL(HS) or 7.5uL(Act5c)　</Td></tr><tr>
<td>　Total　</td><Td>　12uL(HS) or 15uL(Act5c)　</Td></tr>
<td>　Total　</td><Td>　12uL(HS) or 15uL(Act5c)　</Td></tr>
</Table>
</Table>
<br><BR>
<br><BR>
 +
<strong>2-2-14. Transformation of Ligation products</strong>
 +
<br>
 +
Ligation products were transformed into E. coli XL-1 blue.
 +
<br><br>
<h2>September 15th</h2>
<h2>September 15th</h2>
Line 295: Line 376:
-
<strong>*Parts</strong>
+
<strong>1-1-56. Purification of candidate plasmid DNAs</strong>
 +
<br>
 +
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.
<br><br>
<br><br>
 +
Cut check of the candidate pSB1C3-UAS-LacZ DNA, pSB1C3-UAS-EGFP DNA and pSB1C3-G4L4 DNA
 +
<br>
 +
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.
 +
<br><br>
 +
Composition
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　DNA sample　</Td><Td>　1uL　</Td></Tr>
<Tr><Td>　DNA sample　</Td><Td>　1uL　</Td></Tr>
Line 307: Line 396:
</Table>
</Table>
<br><br>
<br><br>
-
 
+
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.
 +
<br><br>
 +
Results
 +
<br>
<img src="https://static.igem.org/mediawiki/2012/d/d3/0915kit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/d/d3/0915kit.png" width="500" height="300">
 +
<br><br>
 +
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.
<br><br>
<br><br>
Line 317: Line 411:
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.
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.
<br><br>
<br><br>
 +
<strong>1-1-58 Preparation of Biobrick part DNAs to submit</strong>
 +
<br>
 +
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℃.
 +
<br><br>
<h2>September 17th</h2>
<h2>September 17th</h2>
Line 326: Line 424:
<br><br>
<br><br>
-
 
+
<strong>1-1-59 Cut check of the Biobrick part DNAs</strong>
-
<strong>*Parts</strong>
+
<br>
 +
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.
<br><br>
<br><br>
-
<Table Border Cellspacing="0">
+
<img src="https://static.igem.org/mediawiki/2012/2/26/0917kit.png" width="500" height="300">
-
<Tr><Td>　DNA sample　</Td><Td>　1uL　</Td></Tr>
+
<br><br>
-
<tr><td>　4 buffer(NEB)　</td><Td>　1uL　</Td></tr>
+
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.
-
<Tr><Td>　EcoRⅠ-HF(NEB)　</Td><Td>　0.2uL　</Td></Tr>
+
-
<Tr><Td>　SpeⅠ(NEB)　</Td><Td>　0.2uL　</Td></Tr><Tr>
+
-
<Td>　100×BSA　</Td><Td>　0.1uL　</Td></Tr><tr>
+
-
<td>　dH<sub>2</sub>O　</td><Td>　7.5uL　</Td></tr><tr>
+
-
<td>　Total　</td><Td>　10uL　</Td></tr>
+
-
</Table>
+
<br><br>
<br><br>
 +
<strong>1-1-60 Submission of the Biobrick parts</strong>
 +
<br>
 +
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.
 +
<br><br>
 +
<strong>1-1-61 Preparation of DNA for transfection into cultured Drosophila cells</strong>
 +
<br>
 +
E. coli carrying pSB1C3-UAS-LacZ and pSB1C3-UAS-EGFP were grown for 16 hours at  37℃.
 +
<br>
-
<img src="https://static.igem.org/mediawiki/2012/2/26/0917kit.png" width="500" height="300">
 
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7">>>>>>>>>>WEEK7</a></div>
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week7">>>>>>>>>>WEEK7</a></div>
</div>
</div>

Revision as of 13:57, 24 September 2012

September 11th


*TNFA and API2

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.

*TNFA and API2

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


*TNFA and API2

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


*TNFA and API2

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


*TNFA and API2

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


*TNFA and API2

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


*TNFA and API2

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℃.

Retrieved from "http://2012.igem.org/Team:KIT-Kyoto/Notebook-week6"