iGEM 2010 — Team Korea_U_Seoul

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# Team:Korea U Seoul/Notebook

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| Contents   * [1 Brain storming & Work notes](#Brain_storming_.26_Work_notes) * [2 Experimental notes](#Experimental_notes)   + [2.1 [Discussion] 2010-08-02 ~ 2010-08-29](#.5BDiscussion.5D_2010-08-02_.7E_2010-08-29)   + [2.2 [ Preparation of competent cells ] 2010-09-01 ~ 2010-09-03](#.5B_Preparation_of_competent_cells_.5D_2010-09-01_.7E_2010-09-03)   + [2.3 [ Transformation efficiency ] 2010-09-04](#.5B_Transformation_efficiency_.5D_2010-09-04)   + [2.4 [ Amplification of BioBrick parts : pSB1A2 and pSB1C3 ] 2010-09-05](#.5B_Amplification_of_BioBrick_parts_:_pSB1A2_and_pSB1C3_.5D_2010-09-05)   + [2.5 [ Genomic DNA extraction ] 2010-09-06](#.5B_Genomic_DNA_extraction_.5D_2010-09-06)   + [2.6 [ Plasmid DNA extraction : pSB1A3 and pSB1C3 ] 2010-09-07](#.5B_Plasmid_DNA_extraction_:_pSB1A3_and_pSB1C3_.5D_2010-09-07)   + [2.7 [ PCR : promoters and reporter genes ] 2010-09-13 ~ 2010-09-16](#.5B_PCR_:_promoters_and_reporter_genes_.5D_2010-09-13_.7E_2010-09-16)   + [2.8 [ Digestion] 2010-09-17](#.5B_Digestion.5D_2010-09-17)   + [2.9 [Chuseok, Korean thanksgiving day] 2010-09-20 ~ 2010-09](#.5BChuseok.2C_Korean_thanksgiving_day.5D_2010-09-20_.7E_2010-09)   + [2.10 [ Ligation & Transformation ] 2010-09-27](#.5B_Ligation_.26_Transformation_.5D_2010-09-27)   + [2.11 [ Confirmation of 1st cloning ] 2010-09-28](#.5B_Confirmation_of_1st_cloning_.5D_2010-09-28)   + [2.12 [ Plasmid DNA extraction : pSB1A2-( PyodA-mAAA-PzntA-RFP) ] 2010-09-29](#.5B_Plasmid_DNA_extraction_:_pSB1A2-.28_PyodA-mAAA-PzntA-RFP.29_.5D_2010-09-29)   + [2.13 [ Digestion ] 2010-10-01 ~ 2010-10-03](#.5B_Digestion_.5D_2010-10-01_.7E_2010-10-03)   + [2.14 [ Confirmation of 2nd cloning ] 2010-10-06](#.5B_Confirmation_of_2nd_cloning_.5D_2010-10-06)   + [2.15 [ Plasmid DNA extraction : pSB1C3-( PyodA-mAAA-PzntA-RFP-ParsR-GFP) ] 2010-10-07](#.5B_Plasmid_DNA_extraction_:_pSB1C3-.28_PyodA-mAAA-PzntA-RFP-ParsR-GFP.29_.5D_2010-10-07)   + [2.16 [ Completion : Heavy-metal detector ] 2010-10-18](#.5B_Completion_:_Heavy-metal_detector_.5D_2010-10-18) |

# **Brain storming & Work notes**

Click on a date to see notes on the meeting & summary of labwork done on that day.

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[http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/10\_June\_2010&action=edit 10] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/11\_June\_2010&action=edit 11] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/12\_June\_2010&action=edit 12] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/13\_June\_2010&action=edit 13] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/14\_June\_2010&action=edit 14] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/15\_June\_2010&action=edit 15] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/16\_June\_2010&action=edit 16] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/17\_June\_2010&action=edit 17] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/18\_June\_2010&action=edit 18] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/19\_June\_2010&action=edit 19] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/20\_June\_2010&action=edit 20] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/21\_June\_2010&action=edit 21] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/22\_June\_2010&action=edit 22] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/23\_June\_2010&action=edit 23] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/24\_June\_2010&action=edit 24] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/25\_June\_2010&action=edit 25] | [http://2010.igem.org/Korea\_U\_Seoul/26\_June\_2010 26] | [http://2010.igem.org/Korea\_U\_Seoul/27\_June\_2010 27] | | [http://2010.igem.org/Korea\_U\_Seoul/28\_June\_2010 28] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/29\_June\_2010&action=edit 29] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/30\_June\_2010&action=edit 30] | | |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | July | | | | | | | | M | T | W | T | F | S | S | |  |  |  | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/1\_July\_2010&action=edit 1] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/2\_July\_2010&action=edit 2] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/3\_July\_2010&action=edit 3] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/4\_July\_2010&action=edit 4] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/5\_July\_2010&action=edit 5] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/6\_July\_2010&action=edit 6] | [http://2010.igem.org/Korea\_U\_Seoul/7\_July\_2010 7] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/8\_July\_2010&action=edit 8] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/9\_July\_2010&action=edit 9] | [http://2010.igem.org/Korea\_U\_Seoul/10\_July\_2010 10] | [http://2010.igem.org/Korea\_U\_Seoul/11\_July\_2010 11] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/12\_July\_2010&action=edit 12] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/13\_July\_2010&action=edit 13] | [http://2010.igem.org/Korea\_U\_Seoul/14\_July\_2010 14] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/15\_July\_2010&action=edit 15] | [http://2010.igem.org/Korea\_U\_Seoul/16\_July\_2010 16] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/17\_July\_2010&action=edit 17] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/18\_July\_2010&action=edit 18] | | [http://2010.igem.org/Korea\_U\_Seoul/19\_July\_2010 19] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/20\_July\_2010&action=edit 20] | [http://2010.igem.org/Korea\_U\_Seoul/21\_July\_2010 21] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/22\_July\_2010&action=edit 22] | [http://2010.igem.org/Korea\_U\_Seoul/23\_July\_2010 23] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/24\_July\_2010&action=edit 24] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/25\_July\_2010&action=edit 25] | | [http://2010.igem.org/Korea\_U\_Seoul/26\_July\_2010 26] | [http://2010.igem.org/Korea\_U\_Seoul/27\_July\_2010 27] | [http://2010.igem.org/Korea\_U\_Seoul/28\_July\_2010 28] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/29\_July\_2010&action=edit 29] | [http://2010.igem.org/Korea\_U\_Seoul/30\_July\_2010 30] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/31\_July\_2010&action=edit 31] | | |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | August | | | | | | | | M | T | W | T | F | S | S | |  |  |  |  |  |  | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/1\_August\_2010&action=edit 1] | | [http://2010.igem.org/Korea\_U\_Seoul/2\_August\_2010 2] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/3\_August\_2010&action=edit 3] | [http://2010.igem.org/Korea\_U\_Seoul/4\_August\_2010 4] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/5\_August\_2010&action=edit 5] | [http://2010.igem.org/Korea\_U\_Seoul/6\_August\_2010 6] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/7\_August\_2010&action=edit 7] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/8\_August\_2010&action=edit 8] | | [http://2010.igem.org/Korea\_U\_Seoul/9\_August\_2010 9] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/10\_August\_2010&action=edit 10] | [http://2010.igem.org/Korea\_U\_Seoul/11\_August\_2010 11] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/12\_August\_2010&action=edit 12] | [http://2010.igem.org/Korea\_U\_Seoul/13\_August\_2010 13] | [http://2010.igem.org/Korea\_U\_Seoul/14\_August\_2010 14] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/15\_August\_2010&action=edit 15] | | [http://2010.igem.org/Korea\_U\_Seoul/16\_August\_2010 16] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/17\_August\_2010&action=edit 17] | [http://2010.igem.org/Korea\_U\_Seoul/18\_August\_2010 18] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/19\_August\_2010&action=edit 19] | [http://2010.igem.org/Korea\_U\_Seoul/20\_August\_2010 20] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/21\_August\_2010&action=edit 21] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/22\_August\_2010&action=edit 22] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/23\_August\_2010&action=edit 23] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/24\_August\_2010&action=edit 24] | [http://2010.igem.org/Korea\_U\_Seoul/25\_August\_2010 25] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/26\_August\_2010&action=edit 26] | [http://2010.igem.org/Korea\_U\_Seoul/27\_August\_2010 27] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/28\_August\_2010&action=edit 28] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/29\_August\_2010&action=edit 29] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/30\_August\_2010&action=edit 30] | [http://2010.igem.org/Korea\_U\_Seoul/31\_August\_2010 31] | | |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | September | | | | | | | | M | T | W | T | F | S | S | |  |  | [http://2010.igem.org/Korea\_U\_Seoul/1\_September\_2010 1] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/2\_September\_2010&action=edit 2] | [http://2010.igem.org/Korea\_U\_Seoul/3\_September\_2010 3] | [http://2010.igem.org/Korea\_U\_Seoul/4\_September\_2010 4] | [http://2010.igem.org/Korea\_U\_Seoul/5\_September\_2010 5] | | [http://2010.igem.org/Korea\_U\_Seoul/6\_September\_2010 6] | [http://2010.igem.org/Korea\_U\_Seoul/7\_September\_2010 7] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/8\_September\_2010&action=edit 8] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/9\_September\_2010&action=edit 9] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/10\_September\_2010&action=edit 10] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/11\_September\_2010&action=edit 11] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/12\_September\_2010&action=edit 12] | | [http://2010.igem.org/Korea\_U\_Seoul/13\_September\_2010 13] | [http://2010.igem.org/Korea\_U\_Seoul/14\_September\_2010 14] | [http://2010.igem.org/Korea\_U\_Seoul/15\_September\_2010 15] | [http://2010.igem.org/Korea\_U\_Seoul/16\_September\_2010 16] | [http://2010.igem.org/Korea\_U\_Seoul/17\_September\_2010 17] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/18\_September\_2010&action=edit 18] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/19\_September\_2010&action=edit 19] | | [http://2010.igem.org/Korea\_U\_Seoul/20\_September\_2010 20] | [http://2010.igem.org/Korea\_U\_Seoul/21\_September\_2010 21] | [http://2010.igem.org/Korea\_U\_Seoul/22\_September\_2010 22] | [http://2010.igem.org/Korea\_U\_Seoul/23\_September\_2010 23] | [http://2010.igem.org/Korea\_U\_Seoul/24\_September\_2010 24] | [http://2010.igem.org/Korea\_U\_Seoul/25\_September\_2010 25] | [http://2010.igem.org/Korea\_U\_Seoul/26\_September\_2010 26] | | [http://2010.igem.org/Korea\_U\_Seoul/27\_September\_2010 27] | [http://2010.igem.org/Korea\_U\_Seoul/28\_September\_2010 28] | [http://2010.igem.org/Korea\_U\_Seoul/29\_September\_2010 29] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/30\_September\_2010&action=edit 30] | | |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | October | | | | | | | | M | T | W | T | F | S | S | |  |  |  |  | [http://2010.igem.org/Korea\_U\_Seoul/1\_October\_2010 1] | [http://2010.igem.org/Korea\_U\_Seoul/2\_October\_2010 2] | [http://2010.igem.org/Korea\_U\_Seoul/3\_October\_2010 3] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/4\_October\_2010&action=edit 4] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/5\_October\_2010&action=edit 5] | [http://2010.igem.org/Korea\_U\_Seoul/6\_October\_2010 6] | [http://2010.igem.org/Korea\_U\_Seoul/7\_October\_2010 7] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/8\_October\_2010&action=edit 8] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/9\_October\_2010&action=edit 9] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/10\_October\_2010&action=edit 10] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/11\_October\_2010&action=edit 11] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/12\_October\_2010&action=edit 12] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/13\_October\_2010&action=edit 13] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/14\_October\_2010&action=edit 14] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/15\_October\_2010&action=edit 15] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/16\_October\_2010&action=edit 16] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/17\_October\_2010&action=edit 17] | | [http://2010.igem.org/Korea\_U\_Seoul/18\_October\_2010 18] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/19\_October\_2010&action=edit 19] | [http://2010.igem.org/Korea\_U\_Seoul/20\_October\_2010 20] | [http://2010.igem.org/Korea\_U\_Seoul/21\_October\_2010 21] | [http://2010.igem.org/Korea\_U\_Seoul/22\_October\_2010 22] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/23\_October\_2010&action=edit 23] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/24\_October\_2010&action=edit 24] | | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/25\_October\_2010&action=edit 25] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/26\_October\_2010&action=edit 26] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/27\_October\_2010&action=edit 27] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/28\_October\_2010&action=edit 28] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/29\_October\_2010&action=edit 29] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/30\_October\_2010&action=edit 30] | [http://2010.igem.org/wiki/index.php?title=Korea\_U\_Seoul/31\_October\_2010&action=edit 31] | |

# **Experimental notes**

## [Discussion] 2010-08-02 ~ 2010-08-29

1. Strategy and overview of iGEM 2010 experiment
[![Kuprojectmain.png](/wiki/images/e/e9/Kuprojectmain.png)](/File%3AKuprojectmain.png)

2. Design of primers

| Primer | Sequence ( 5’ → 3’ ) |
| --- | --- |
| PyodA(*Eco*RI)\_F | CCGGAATTCCTTCATATTGCCGACAAAGTACG |
| mAAA(*Spe*I)\_R | GGACTAGTTTATCACAGGGGCCGTCCG |
| PzntA(*Xba*I)\_F | GCTCTAGACGTCCGCTCGCTGTATCTC |
| RFP(*Pst*I)\_R | AACTGCAGCGGCCGCTACTAGTTTATTAAGCACCGGTGGAGTGA |
| ParsR(*Xba*I)\_F | GCTCTAGACCAACTCAAAATTCACACCTATTAC |
| GFP(*Pst*I)\_R | AACTGCAGTTAAGGCCTTTTGTATAGTTCATCC |

## [ Preparation of competent cells ] 2010-09-01 ~ 2010-09-03

1. Inoculation of *E. coli* DH5α and *E. coli* BL21(DE3) to 3mL LB broth

2. Preparation of 200mL 2x LB broth, TSS solution and LB plates with ampicillin(100μg/mL) and chloramphenicol(25μg /mL), respectively

3. Inoculation of subcultured *E. coli* to 200mL 2x LB borth

4. Preparation of competent cells by CSBL laboratory protocol

5. Transformation of pUC19 plasmid(10ng/μL) to competent cells for transformation efficiency check

## [ Transformation efficiency ] 2010-09-04

| Strain | Number of colonies (colonies/μg DNA) |
| --- | --- |
| *E. coli* DH5α | Number of colonies (colonies/μg DNA) |
| *E. coli* BL21(DE3) | 1.5 x 105 |

## [ Amplification of BioBrick parts : pSB1A2 and pSB1C3 ] 2010-09-05

1. Confirmed location : pSB1A2-BBa\_E0040 (2010 Kit plate 1/ 14K) and pSB1C3-BBa\_J04450 (2010 Kit plate 1/ 3A)

2. 20uL suspension by autoclaved distilled water

3. 3uL transformation to *E. coli* DH5α

4. Plating to LB(Amp100), LB(Cm25)

## [ Genomic DNA extraction ] 2010-09-06

1. Inoculation for plasmid DNA purification

2. *E. coli* K12 genomic DNA extraction by AccuPrep® Genomic DNA Extraction Kit

3. Confirmation of genomic DNA by agarose gel electrophoresis (Figure 1)

4. Quantification of DNA concentration by NanoDrop : 137.5ng/μL

[![](/wiki/images/2/25/Real_figure_1.jpg)](/File%3AReal_figure_1.jpg)

figure1. lane1;M-lane2;*E. coli* K12 genomic DNA extraction

br>

## [ Plasmid DNA extraction : pSB1A3 and pSB1C3 ] 2010-09-07

1. Plasmid miniprep by LaboPass™ Plasmid Mini (Plasmid DNA purification kit)

2. Confirmation of extracted plasmids by agarose gel electrophoresis (Figure 2)

3. Quantification of DNA concentration by NanoDrop

[![](/wiki/images/9/9c/Figure1_%EC%8B%A4%ED%97%98.png)](/File%3AFigure1_%EC%8B%A4%ED%97%98.png)

figure2.**lane1;M-lane2;pSB1A2-lane3;pSB1C3**

## [ PCR : promoters and reporter genes ] 2010-09-13 ~ 2010-09-16

1. PCR : PyodA-mAAA, PzntA-RFP(BBa\_E1010) and ParsR-GFP(BBa\_E0040)

| Reagent | Volume (μL) |
| --- | --- |
| 2.5mM dNTP | 3 |
| 10x buffer | 5 |
| Plasmid template (20ng/μL) | 2 |
| Primers (10pmole/μL) | 4 |
| α-Taq DNA polymerase (5U/μL) | 0.5 |
| D.W. | 35.5/total=50 |
| 95˚C(2’)-[95˚C(20”)-55˚C(20”)-72˚C(2’)]30-72˚C(5’)-4˚C |

2. Confirmation of PCR products by agarose gel electrophoresis (Figure 3)

3. Purified PCR products

4. Quantification of DNA concentration by NanoDrop

[![](/wiki/images/b/b5/Figure2_%EC%8B%A4%ED%97%98.png)](/File%3AFigure2_%EC%8B%A4%ED%97%98.png)

figure3.lane1;M-lane2;(PyodA-mAAA)-lane3;(Pznt-RFP)-lane4;(ParsR-GFP)

## [ Digestion] 2010-09-17

1. Digestion of PCR products and pSB1A2

:   1) PyodA-mAAA : *Eco*RI and *Spe*I

:   2) PzntA-RFP : *Xba*I and *Pst*I

:   3) pSB1A3 : *Eco*RI and *Pst*I

| Reagent | Volume (μL) |
| --- | --- |
| DNA (about 30ng/μL) | 30 |
| 10x NEB buffer 2 | 5 |
| BSA (10mg/mL) | 0.5 |
| Appropriate 1st and 2nd restriction enzymes | 2 (each 1) |
| D.W. | 12.5 / total = 50 |
| Completely digestion at 37˚C for 2 hours (at least) and stop at 80˚C for 20min |

2. Confirmation of digested products by agarose gel electrophoresis (Figure 4)

3. Quantification of DNA concentration by NanoDrop

[![](/wiki/images/2/23/4_Figure3_%EC%8B%A4%ED%97%98.png)](/File%3A4_Figure3_%EC%8B%A4%ED%97%98.png)

figure4.M pSB1A2(*Eco*RI/*Pst*I) PyodA-mAAA(*Eco*RI/*Spe*I) PzntA-RFP(*Xba*I/*Pst*I)

## [Chuseok, Korean thanksgiving day] 2010-09-20 ~ 2010-09

## [ Ligation & Transformation ] 2010-09-27

1. Ligation of each parts : PyodA-mAAA, PzntA-RFP and pSB1A2

| Reagent | Volume (μL) |
| --- | --- |
| 10x T4 DNA ligase reaction buffer | 2 |
| T4 DNA ligase | 2 |
| Each of the digests | 2 + 2 + 2 = 8 |
| D.W. | 8 / total = 20 |
| Incubation at room temperature for 30min and stop at 80˚C for 20min |

2. Transformation to *E. coli* DH5α

## [ Confirmation of 1st cloning ] 2010-09-28

1. Check : the color of colonies **(pSB1A2 : green, recombinant plasmid : white)**

2. Inoculation of white colonies to 3mL LB(Amp100)

## [ Plasmid DNA extraction : pSB1A2-( PyodA-mAAA-PzntA-RFP) ] 2010-09-29

1. Plasmid DNA purification by LaboPass™ Plasmid Mini

2. Confirmation of extracted plasmids by agarose gel electrophoresis (Figure 5)

3. Recombinant plasmid sequencing by COSMO GeneTech

[![](/wiki/images/3/38/5_Figure4_%EC%8B%A4%ED%97%98.png)](/File%3A5_Figure4_%EC%8B%A4%ED%97%98.png)

figure5. lane1;M-lane2; (pSB1A2)-[PyodA-mAA-Pznt-RFP] #1,2,3

## [ Digestion ] 2010-10-01 ~ 2010-10-03

1. Check : recombinant plasmid sequence
2. Selection of correct clones
3. Digestion of PCR products(ParsR-GFP) and pSB1C3

:   1) PyodA-mAAA-PzntA-RFP : *Eco*RI and *Spe*I
:   2) ParsR-GFP : *Eco*RI and *Spe*I
:   3) pSB1C3 : *Eco*RI and *Pst*I

| Reagent | Volume (μL) |
| --- | --- |
| DNA (about 30ng/μL) | 30 |
| 10x NEB buffer 2 | 5 |
| BSA (10mg/mL) | 0.5 |
| Appropriate 1st and 2nd restriction enzymes | 2 (each 1) |
| D.W. | 12.5 / total = 50 |
| Completely digestion at 37˚C for 2 hours (at least) and stop at 80˚C for 20min |

4. Confirmation of digested products by agarose gel electrophoresis (Figure 6)

[File:Figure6 aaa.png](/File%3AFigure6_aaa.png "File:Figure6 aaa.png")

figure6. lane1; M-lane2; (PyodA-mAAA-PzntA-RFP(EcoRI/SpeI))-lane3; (ParsR-GFP(XbaI/SpeI))-lane4; (pSB1C3(EcoRI/PstI))

5. Quantification of DNA concentration by NanoDrop

6. Ligation of each parts : PyodA-mAAA-PzntA-RFP, ParsR-GFP and pSB1C3

| Reagent | Volume (μL) |
| --- | --- |
| 10x T4 DNA ligase reaction buffer | 2 |
| T4 DNA ligase | 2 |
| Each of the digests | 2 + 2 + 2 = 8 |
| D.W. | 8 / total = 20 |
| Incubation at room temperature for 30min and stop at 80˚C for 20min |

7. Transformation to *E. coli* DH5α

## [ Confirmation of 2nd cloning ] 2010-10-06

1. Check : the color of colonies (pSB1C3 : red, recombinant plasmid : white)

2. Inoculation of white colonies to 3mL LB(Amp100)

## [ Plasmid DNA extraction : pSB1C3-( PyodA-mAAA-PzntA-RFP-ParsR-GFP) ] 2010-10-07

1. Plasmid DNA purification by LaboPass™ Plasmid Mini

2. Confirmation of extracted plasmids by agarose gel electrophoresis (Figure 7)

3. Recombinant plasmid full-sequencing by COSMO GeneTech

[![](/wiki/images/0/0d/Figure7_%EC%8B%A4%ED%97%98.png)](/File%3AFigure7_%EC%8B%A4%ED%97%98.png)

figure7. lane1;M-lane2; (psB1A2) Heavy-metal detector #1~3

## [ Completion : Heavy-metal detector ] 2010-10-18

1. Check : recombinant plasmid sequence

2. Selection of correct clones

3. Transformation to *E. coli* BL21(DE3) for expression test

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