From 2012.igem.org

(Difference between revisions)

Latest revision as of 01:00, 27 October 2012

KAIST Korea 2012 iGEM

HOME
- Introduction
- iGEM
TEAM
- Official Team Profile
- Members
- Gallery
DATA
- Description
- Bio Bricks

PROJECT
- Overview
- Results
- Future Plan
- Modeling
NOTEBOOK
- Labnote
- Protocol
PLUS
- Safety
- Human Practice
- Attribution
- Sponsors

2012 KAIST Korea

Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
Facebook : www.facebook.com/KAISTiGEM2012

Notebook : Labnote-July

Labnote

July

July 1^st 2012

　PACKMAN

Moth 0109, 1202 TOPO cloning colony PCR

Results

We did colony PCR for the 5 colony of TOPO cloned samples, but there was no band, which means TOPO cloning has failed for these two genes.

Back to the Calendar

July 2^nd 2012

　PACKMAN

fdnG deletion PCP20 curing

fdnG deletion PCP20 prep, single cut

Back to the Calendar

July 3^rd 2012

　PACKMAN

Moth_0109, 1202 PCR

As TOPO cloning of Moth_0109 and 1202 gene failed, we ran one more PCR to prepare for TOPO cloning.

Results

For each of the two genes, correct size of oligonucleotide exists in the PCR product. We noticed that there is an other restriction enzyme site in the Moth 0109, 1202. It means that it’s impossible to reuse TOPO vector. We got other vector which has same pBAD promoter with TOPO cloning. Also we redesigned the primer for Moth 0109, 1202.

pre-culture of Moth_1197 and 1198 inserted MG1655 cell.

We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.

Back to the Calendar

July 4^th 2012

　PACKMAN

Induction of Moth_1197, Moth_1198 gene (sampling) and running SDS-PAGE gel.

We induced Moth_1197 and Moth_1198 gene in pTrcHis2A and pBAD vector with various IPTG and L-arabinose conditions: IPTG 0.5mM, 1mM and Arabinose 10mM, 30mM (0.02% and 0.2%). Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.

Back to the Calendar

July 5^th 2012

　PACKMAN

fdhF Knockout PCR ( negative and knockout) and PCP20?

Results

Moth_1197-pBAD/TOPO expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared slightly above 42kDa. This means that protein has N and C terminal tag, of which the size is 13kDa and 3kDa each.

28.61kDa + N-term Thioredoxin 13kDa + C-term V5, 6xHis 3kDa = 44.61kDa

Therefore, the product protein locates slightly above 42kDa ladder.

Moth_1197-pTrcHis2A expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared between 24kDa and 35kDa. This means that protein has C terminal tag,

28.61kDa + 2.14kDa = 30.75kDa

The band that does not exist on negative sample is roughly on this size. Therefore, we can say that this protein is also expressed on pTrcHis2A vector. However, we decided to use pBAD/TOPO vector because it has expressed the protein more significantly.

Moth_1198-pBAD/TOPO expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. But the band was not certain on the gel image.

Moth_1198-pTrcHis2A expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. The band of correct size appeared on the soluble fraction of each sample.

Back to the Calendar

July 6^th 2012

No Special Event!

Back to the Calendar

July 7^th 2012

No Special Event!

Back to the Calendar

July 8^th 2012

　PACKMAN

Moth 0109, 1202 colony PCR for TOPO cloning to TOP10

Results

1202 colony PCR, The size of bands are correct. We succeed Moth 0109, 1202 gene cloning.

Back to the Calendar

July 9^th 2012

　PACKMAN

fdoG knockout confirm PCR

Results

Moth 0109 and Moth 1202 electro transformation into MG1655 cell

Procedure

We did mini-prep to get the vectors which has Moth 0109, 1202 each from TOP10. We did electroporation to MG1655.

Back to the Calendar

July 10^th 2012

　PACKMAN

piBR181 vector arrived

piBR181 vector is known to accommodate oligonucleotide over 100kb. This vector was made from commercial pET-28a (+) vectors to accommodate standardized Biobricks parts. The work proceeded by designing a 181 bp long synthetic DNA fragment for efficient construction of an expression vector for monocistronic assembly of genes provided that each contains its own promoter, operator, ribosome-binding site and transcriptional terminator. A 181 bp long ds DNA construct was designed and ligated to pET-28a (+) (5369 bp) at BglII and XhoI restriction sites to generate piBR181 (5301 bp) expression vector replacing original bio-parts. The newly constructed vector contains SpeI-HindIII single cloning restriction site between RBS and TT sites however, BglII and BamHI/XhoI restriction sites are present before promoter and after TT sites for multi-monocistronic operon assembly.

We got this standardized vector and vector map from Professor Jae Kyung, Song and it arrived today.

▶▶Click to download Original document of piBR181 vector

Moth 0109 transformed cell confirm PCR

Results

There was no band appeared on the gel, which means that we don’t have any colony with Moth 0109 gene.

Moth 1202 transformed cell confirm PCR

Results

Band size is smaller than Moth 1202. It means we failed to transform Moth 1202 gene in to MG1655.

Back to the Calendar

July 11^th 2012

No Special Event!

Back to the Calendar

July 12^th 2012

　PACKMAN

pre-culture of Moth_1202, 2312 and 2315 inserted MG1655 cell.

We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.

Back to the Calendar

July 13^th 2012

　PACKMAN

Induction of Moth_1202, 2312 and 2314 gene (sampling) and running SDS-PAGE gel.

We induced Moth_1202, 2312 and 2314 gene in pTrcHis2A and pBAD vector with various IPTG and L-arabinose conditions: IPTG 0.5mM, 1mM and Arabinose 10mM, 30mM (0.02% and 0.2%). Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.

Moth_0109 transformed cell confirm PCR – second trial

Results

There was no band appeared on the gel, which means that we don’t have any colony with Moth_0109 gene.

Back to the Calendar

July 14^th 2012

　PACKMAN

piBR181 vector single cut

piBR181 vector was cut with RE to check the size of this vector.

Results

Moth_1202-pTrcHis2Aexpression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72kDa protein, bifunctional acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. Although the band appeared slightly between 75kDa and 90kDa, we were not certain that this band is from Moth 1202 gene expression. This band also exists on the negative sample. Therefore, we concluded that expression of 1202 gene on pTrcHis2A vector has failed.

Moth_2312-pBAD/TOPO expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 98.60kDa protein formate dehydrogenase subunit alpha. Although the band significantly appeared slightly above 42kDa, this was not the correct size of protein. Therefore, we concluded that expression of 2312 gene on pBAD/TOPO vector has failed.

Moth_2314-pBAD/TOPO expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 78.07kDa protein formate dehydrogenase beta subunit. The band was not certain on the gel image. Therefore, we concluded that expression of 2314 gene on pBAD/TOPO vector has failed.

Moth_2314- pTrcHis2A expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 78.07kDa protein formate dehydrogenase beta subunit. The band was not certain on the gel image. Therefore, we concluded that expression of 2314 gene on pTrcHis2A vector has failed.

pre-culture of Moth_1202, 1198 and 0109 inserted MG1655 cell.

We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.

Back to the Calendar

July 15^th 2012

　PACKMAN

Induction of Moth_1202, 1198 and 0109 gene (sampling) and running SDS-PAGE gel.

We induced Moth-1202, 1198 and 0109 gene in pTrcHis2A and pBAD vector with various IPTG and L-arabinose conditions: IPTG 0.5mM, 1mM and Arabinose 10mM, 30mM (0.02% and 0.2%). Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.

　Flip Flop

Project design

Results

The att B and att P sequences are recognition site of bacteriophage Bxb1 integrase. To prove our concept, we designed two plasmid called pPoC (proof of concept plasmid) and pPoCpi (proof of concept plasmid, promoter inverted). The pPoC insert and pPoCpi insert are cloned into pSB1C3 to become pPoC and pPoCpi.

The three sequences - Bacteriophage Bxb1 integrase, sequences between BBa_E1010 and BBa_E0040 of the inserts, pPoC insert and pPoCpi insert – are synthesized through gene synthesis service provided by BIONEER.

Back to the Calendar

July 16^th 2012

　PACKMAN

Moth_1202-pBAD/TOPO expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72kDa protein, bifunctional acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. Although the band appeared slightly above 75kDa, we were not certain that this band is from Moth 1202 gene expression. We thought that band of size 81.72kDa should be more biased towards 90kDa band. Therefore, we concluded that expression of 1202 gene on pBAD vector has failed.

Moth_1198-pBAD/TOPO expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. The band significantly appeared slightly above 42kDa. This means that protein has N terminal tag, of which the size is 13kDa.

35.08kDa + N-term Thioredoxin 13kDa = 48.08kDa

Therefore, the product protein locates slightly above 42kDa ladder

Moth_1198-pTrcHis2A expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72kDa protein, bifunctional acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. Although the band appeared slightly above 75kDa, we were not certain that this band is from Moth 1202 gene expression. We thought that band of size 81.72kDa should be more biased towards 90kDa band. Therefore, we concluded that expression of 1202 gene on pBAD vector has failed.

Moth_0109-pTrcHis2A expression check (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. The band is not significant, but appeared slightly above 54kDa marker in the 37℃ sample.

Back to the Calendar

July 17^th 2012

　Flip Flop

Part preparation

We selected some parts, needed for this project, from the partregistry. We requested 10 parts which are not included in distribution kit.

Results

Also, we designed primers for constructing ‘proof of concept plasmid’.

Transformation of parts into TOP10

Parts:

BBa_E0040 (wild-type GFP); AmpR
BBa_E1010 (mRFP); KanR

Competent cell: TOP10
Method: heat-shock

Results

BBa_E0040: Colony formed, will be checked by restriction cut of purified plasmid DNA.
BBa_E1010: No colony

Discussions

BBa_E1010 will be transformed into MG1655 by electro-transformation to make sure.

Back to the Calendar

July 18^th 2012

　Flip Flop

PCR amplification of parts

Parts:

BBa_E0040(720bp) with primer GFP-F and Plasmid1-R: 740bp
BBa_E1010(681bp) with primer Plasmid1-F and mRFP-R: 701bp

Template: distributed parts 1uL

Results

Gel electrophoresis

PCR purification:

BBa_E0040: 48.2ng/uL (purity 1.88)
BBa_E1010: 104.3ng/uL (purity 1.88)

Discussions

The bands of both parts showed right size and yield is considerable.

Back to the Calendar

July 19^th 2012

　Flip Flop

BBa_E0040 transformants plasmid DNA mini-prep & enzyme cut check

Strain: TOP10 - BBa_E0040

BBa_E0040 (2799bp): GFP (720bp) cloned into pSB1A2 (2079bp)

Restriction enzyme digestion conditions

Incubated in 37℃ for 1hr

Results

DNA Concentration measurement:

Colony #1: 161.3ng/uL (purity 1.74)
Colony #2: 140.5ng/uL (purity 1.51)
Colony #3: 250.6ng/uL (purity 1.83)

Digested DNA gel electrophoresis

Discussions

The bands of three parts showed size little bit larger than desired size. This might be due to additional bases, prefix and suffix, attached during partregistry assembly process. Also, yield is considerable.

BBa_E1010 electroporation into MG1655

Part: BBa_E1010 (mRFP); KanR
Strain: MG1655

Back to the Calendar

July 20^th 2012

　Flip Flop

MG1655-BBa_E1010 colony inoculation

Three colonies were formed. All of them were inoculated in 3mL of LB with 1% of kanamycin.

Back to the Calendar

July 21^st 2012

　Flip Flop

BBa_E1010 transformants plasmid DNA mini-prep & enzyme cut check

Strain: MG1655
BBa_E1010 (5107bp): mRFP (681bp) cloned into pSB2K3 (4426bp)

Restriction enzyme digestion conditions

Incubated in 37℃ for 1hr

Results

No band appeared

Back to the Calendar

July 22^nd 2012

　PACKMAN

pre-culture of MG 1655 cell transformed with Moth 1202 gene inserted pTrcHis2A and pBAD/mycHisC vector.

We did pre-culture of both pTrcHis2A 1202 and pBAD/mycHisC transformed cell into 5ml LB and incubated on 37℃ for 16 hours.

　Flip Flop

BBa_E1010 transformants plasmid DNA mini-prep

Results

Colony #1: 26.2ng/uL (purity 2.47)

Discussions

The pSB2K3 is high copy plasmid only if induced by IPTG. Without induction, it is low copy vector. For higher yield, we induce cells with IPTG tomorrow.

Primer design for Cre, FLP cloning

Restriction site is suit for both pTrcHis2a and pBADmychisC vector MCS.

Back to the Calendar

July 23^rd 2012

　PACKMAN

Moth 0109 vector double cut check

Restriction and buffers are mixed in the ratio as shown below.

Results

We checked the size of each broken fragments of vector, and found each bands appear about 1.6kb. After checking correct size of fragment, we sent mini-prepped vectors for sequencing.

Induction of Moth_1202 gene (sampling) and running SDS-PAGE gel.

Last time we induced the samples in 0.5mM, 1mM IPTG and 10mM arabinose conditions, but there were no significant band on the gel. So we induced Moth_1202 gene in pTrcHis2A and pBAD vector with more larger concentration of IPTG and arabinose conditions: IPTG 2mM, 3mM, 4mM and Arabinose 10mM, 30mM, 40mM. Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.

Back to the Calendar

July 24^th 2012

　PACKMAN

Expression check of Moth_1202 on pBAD vector (Result)

Results

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72 kDa protein, acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. The band appeared significantly on the correct size for pTrcHis2A vector lanes.

　Flip Flop

Cre and Flp recombinase gene amplification and purification

Cre recombinase (1032bp) and Flp recombinase (1292bp) were amplified from BBa_J61047 and pCP20 respectively using HotStarTaq DNA polymerase. Amplified genes were purified by PCR purification kit.

Results

Cre: 175.6 ng/uL (purity: 1.89)
Flp: 105.3 ng/uL (purity: 1.88)

Synthesized gene transformation

Synthetic constructs of pPoC insert and pPocpi insert were arrived. They were TA cloned into pGEM-B1 vector. We chemically transformed the vectors to DH5α as recommended from BIONEER.

Back to the Calendar

July 25^th 2012

　PACKMAN

Sequencing result of Moth_0109

We analyzed sequencing result from Macrogen using Multialin program.

Results

Cite: "Multiple sequence alignment with hierarchical clustering". F. CORPET, 1988, Nucl. Acids Res., 16 (22), 10881-10890

　Flip Flop

Colony PCR

Colony PCR of colonies from BBa_E0040 (720bp) and BBa_E1010 (681bp) master plates of July 17^th.

Results

Inoculation of cells having P1-pGEM and PI-pGEM

Inoculated the transformed cells into 3mL of LB broth with 1% of ampicillin.

Back to the Calendar

July 26^th 2012

　PACKMAN

Vector mini-prep for all genes except 0109 and FDH.

Results

colony PCR of Moth_0109 gene

Results

　Flip Flop

vector preparation and transformation check

MG1655 strains that carrying pTrcHis2A (4.4kb) and pBAD/Myc-HisC (4.1kb) were inoculated. We extracted plasmid DNA from the cells.

P1-pGEM and PI-pGEM plasmids were also extracted.

All the plasmids were checked with restriction enzyme cut.

pTrcHis2A and pBAD/Myc-HisC with EcoRI
P1-pGEM and PI-pGEM with HindIII

Results

Back to the Calendar

July 27^th 2012

　PACKMAN

Primer design for Gibson assembly

Results

According to the Gibson assembly kit from NEB,

PCR amplification of fragments for Gibson assembly

Results

PCR amplification result was successful except for 1199, 1191 genes; bands appear at the correct size. Correct size for each fragments are as below.

1201 : 1.3 + 0.3 = 1.6kb
1203 :
1204
1197
1198
1199
1191
1516

　Flip Flop

pPoC insert and pPocpi insert construction: Trial1

pPoc insert consists of BBa_E1010, P1 and BBa_E0040 and pPoC insert consists of BBa_E1010, PI and BBa_E0040 were constructed using Overlapping Extension(OE) PCR.

Template preparation

OE PCR

We mixed each template with equimolar amount.

Results

Failed to construct the insert sequences of pPoC and pPoCpi.

Discussions

We missed adding pfu-X polymerase when amplifying templates and OE PCR. We think that is the reason why we failed to construct inserts.

Back to the Calendar

July 28^th 2012

　PACKMAN

pre-culture of pTrcHis2A-Moth0109 vector inserted MG1655 cell.

We did pre-culture of pTrcHis2A 0109 transformed cell into 5ml LB and incubated on 37℃ for 16 hours.

　Flip Flop

pPoC insert and pPocpi insert construction: Trial2

Template preparation

pPoC & pPoCpi promoter segment(214bp)

OE PCR

→ Gel extracted

Discussions

When performing OE PCR, adding forward and reverse primers of full fragment at the first time make the yield higher than without adding primers.

Back to the Calendar

July 29^th 2012

　PACKMAN

PCR amplification of pBAD - Moth_1191 & 1199

We amplified target genes using PCR. Total reaction volume was 50uL and the elongation time was about 1kb/min.

Results

second trial of Moth_1199, 1191 amplification

This was the second trial of amplifying Moth 1199 and 1191, but we found no band on the gel. So, we decreased annealing temperature down to 51℃, but this time we also couldn’t find one.

pre-culture of cell for vector miniprep - Moth_1191 & 1199

As there might be errors in vector miniprep step, we decided to pre-culture the cell once more.

Induction of Moth_0109 gene (sampling) and running SDS-PAGE gel.

We induced Moth-0109 gene in pTrcHis2A vector with different IPTG conditions and temperature: 0.5mM, 1mM and 37℃, 30℃. And then, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.

　Flip Flop

pPoC, pPoCpi Cloning

Cloning Conditions – iGEM recommended protocol

Restriction enzyme digestion

Enzyme pre-mix – 37℃ 1hr, 80℃ 20min

Digestion mixture – 37℃ 1hr, 80℃ 20min

Ligation - 16℃ 1hr, 80℃ 20min

Transformed into DH5α chemically competent cell.

Back to the Calendar

July 30^th 2012

　PACKMAN

Expression optimization of Moth_0109 gene on pTrcHis2A (Result)

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94 kDa protein, formate tetrahydrofolate ligase.

Results

Discussion

PCR amplification of Moth_1191, 1199 gene (3rd and 4th trial)

We did PCR amplification of Moth_1191 and 1199 gene with original TOPO primer and Gibson primer.

Results

We thought that there might be some problem with vector preparation, so we tried mini-prep once more, and we got no band. Also, we tried colony PCR of 1191 and 1199 gene with Gibson primers, which has failed.

　Flip Flop

Ligation product plating

Results

No colony appeared

Discussions

Maybe iGEM recommended protocol is inappropriate or we have to increase our insert DNA concentration.

Primer design for pPoC and pPoCpi

We decided not to use linearized plasmid backbone. Thus we designed primers which have EcoRI and PstI as its restriction site.

Back to the Calendar

July 31^st 2012

　PACKMAN

PCR amplification of Moth_1191, 1199 gene (5th trial)

We did PCR amplification of Moth_1191 and 1199 gene with original TOPO primer and Gibson primer.

Results

We can see that only the samples with TOPO primers had the correct size of oligonucleotide. Therefore, there might be some problems with forward primers.

pre-culture of pBAD/mycHisC-Moth0109 vector inserted MG1655 cell.

We did pre-culture of pBAD/mycHisC 0109 transformed cell into 5ml LB and incubated on 37℃ for 16 hours.

Back to the Calendar

Kaist Footer

@@ Line 35: / Line 35: @@
 		z-index: 2;
 	}
+	#linegray
+	{
+		background-color:transparent;
+		height:20px;
+		width:670px;
+		padding: 10px 0px 10px 0px;
+	}
 	#kaistcontent #starter-grad
 	{
@@ Line 186: / Line 193: @@
 			<section id="1">
 				<div class="date">July 1<sup>st</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title"><i>Moth 0109</i>, <i>1202</i> TOPO cloning colony PCR</div>
@@ Line 211: / Line 220: @@
 			<section id="2">
 				<div class="date">July 2<sup>nd</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">fdnG deletion PCP20 curing</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">fdnG deletion PCP20 prep, single cut</div>
@@ Line 226: / Line 235: @@
 				<div class="date">July 3<sup>rd</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title"><i>Moth_0109</i>, <i>1202</i> PCR</div>
@@ Line 246: / Line 256: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">pre-culture of <i>Moth_1197</i> and <i>1198</i> inserted MG1655 cell. </div>
@@ Line 262: / Line 272: @@
 			<section id="4">
 				<div class="date">July 4<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Induction of <i>Moth_1197</i>, <i>Moth_1198</i> gene (sampling) and running SDS-PAGE gel.</div>
@@ Line 278: / Line 289: @@
 			<!--제목에 물음표는 뭘까;;?-->
 				<div class="date">July 5<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">fdhF Knockout PCR ( negative and knockout) and PCP20?</div>
@@ Line 289: / Line 301: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_1197</i>-pBAD/TOPO expression check (Result)</div>
@@ Line 307: / Line 319: @@
 					</br>
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_1197</i>-pTrcHis2A expression check (Result)</div>
@@ Line 326: / Line 338: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_1198</i>-pBAD/TOPO expression check (Result)</div>
@@ Line 343: / Line 355: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_1198</i>-pTrcHis2A expression check (Result)</div>
@@ Line 364: / Line 376: @@
 			</section>
 			<section id="6">
 				<div class="date">July 6<sup>th</sup> 2012</div></br>
 				<div id="content_note" >
@@ Line 386: / Line 399: @@
 			<section id="8">
 				<div class="date">July 8<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title"><i>Moth 0109</i>, <i>1202</i> colony PCR for TOPO cloning to TOP10</div>
@@ Line 410: / Line 425: @@
 			<section id="9">
 				<div class="date">July 9<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">fdoG knockout confirm PCR</div>
@@ Line 420: / Line 436: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth 0109</i> and <i>Moth 1202</i> electro transformation into MG1655 cell</div>
@@ Line 439: / Line 455: @@
 			<section id="10">
 				<div class="date">July 10<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">piBR181 vector arrived</div>
@@ Line 460: / Line 477: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth 0109</i> transformed cell confirm PCR</div>
@@ Line 478: / Line 495: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth 1202</i> transformed cell confirm PCR</div>
@@ Line 512: / Line 529: @@
 			<section id="12">
 				<div class="date">July 12<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">pre-culture of <i>Moth_1202</i>, <i>2312</i> and <i>2315</i> inserted MG1655 cell. </div>
@@ Line 527: / Line 545: @@
 			<section id="13">
 				<div class="date">July 13<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Induction of <i>Moth_1202</i>, <i>2312</i> and <i>2314</i> gene (sampling) and running SDS-PAGE gel. </div>
@@ Line 537: / Line 556: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_0109</i> transformed cell confirm PCR – second trial</div>
@@ Line 559: / Line 578: @@
 			<section id="14">
 				<div class="date">July 14<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">piBR181 vector single cut</div>
@@ Line 574: / Line 594: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_1202</i>-pTrcHis2Aexpression check (Result)</div>
@@ Line 595: / Line 615: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_2312</i>-pBAD/TOPO expression check (Result)</div>
@@ Line 617: / Line 637: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_2314</i>-pBAD/TOPO expression check (Result)</div>
@@ Line 640: / Line 660: @@
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_2314</i>- pTrcHis2A expression check (Result)</div>
@@ Line 662: / Line 682: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">pre-culture of <i>Moth_1202</i>, <i>1198</i> and <i>0109</i> inserted MG1655 cell. </div>
@@ Line 678: / Line 698: @@
 				<div class="date">July 15<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Induction of <i>Moth_1202</i>, <i>1198</i> and <i>0109</i> gene (sampling) and running SDS-PAGE gel. </div>
@@ Line 685: / Line 706: @@
 					<span id="little">We induced <i>Moth-1202</i>, <i>1198</i> and <i>0109</i> gene in pTrcHis2A and pBAD vector with various IPTG and L-arabinose conditions: IPTG 0.5mM, 1mM and Arabinose 10mM, 30mM (0.02% and 0.2%). Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.</span>
 					</br></br>
+				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">Project design</div>
+				<div id="content_note" >
+					<b>Results</b></br></br>
+					<div align="center"><img id="figure" alt="2_0715Fig1" src="https://static.igem.org/mediawiki/2012/0/06/KAIST_2nd_07152012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+					<span id="little">The <i>att B</i> and <i>att P</i> sequences are recognition site of bacteriophage Bxb1 integrase. To prove our concept, we designed two plasmid called pPoC (proof of concept plasmid) and pPoCpi (proof of concept plasmid, promoter inverted). The pPoC insert and pPoCpi insert are cloned into pSB1C3 to become pPoC and pPoCpi.  </br></br>The three sequences - Bacteriophage Bxb1 integrase, sequences between BBa_E1010 and BBa_E0040 of the inserts, pPoC insert and pPoCpi insert – are synthesized through gene synthesis service provided by BIONEER.
+					</span>
+					</br>
 				</div>
@@ Line 694: / Line 735: @@
 			<section id="16">
 				<div class="date">July 16<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title"><i>Moth_1202</i>-pBAD/TOPO expression check (Result)</div>
@@ Line 715: / Line 757: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_1198</i>-pBAD/TOPO expression check (Result)</div>
@@ Line 736: / Line 778: @@
 					</br>
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_1198</i>-pTrcHis2A expression check (Result)</div>
@@ Line 757: / Line 799: @@
 					</br>
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title"><i>Moth_0109</i>-pTrcHis2A expression check (Result)</div>
@@ Line 779: / Line 821: @@
 			</section>
 			<section id="17">
 				<div class="date">July 17<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">Part preparation</div>
 				<div id="content_note" >
-					<span id="little">No Special Event!</span>
+					<span id="little">We selected some parts, needed for this project, from the partregistry. We requested 10 parts which are not included in distribution kit.</span>
+					</br></br>
+					<b>Results</b></br></br>
+					<div align="center"><img id="figure" alt="2_0717Fig1" src="https://static.igem.org/mediawiki/2012/5/55/KAIST_2nd_07172012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
 					</br>
+					<span id="little">  Also, we designed primers for constructing ‘proof of concept plasmid’.</span>
+					</br></br>
+					<div align="center"><img id="figure" alt="2_0717Fig2" src="https://static.igem.org/mediawiki/2012/0/09/KAIST_2nd_07172012_Fig2.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+				</div>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
+				<div class="note-title">Transformation of parts into TOP10</div>
+				<div id="content_note" >
+					<span>
+					Parts:</br>
+					<ul style="list-style-type:square">
+					<li>BBa_E0040 (wild-type GFP); <i>AmpR</i></li>
+					<li>BBa_E1010 (mRFP); <i>KanR</i></li>
+					</ul>
+					Competent cell: TOP10</br>
+					Method: heat-shock</br>
+					</span>
+					</br></br>
+					<b>Results</b></br></br>
+					<span id="little">
+					<ul style="list-style-type:square">
+					<li>BBa_E0040: Colony formed, will be checked by restriction cut of purified plasmid DNA.</br></li>
+					  <li>BBa_E1010: No colony</span></br></br></li>
+					</ul>
+					<b>Discussions</b></br></br>
+					<span id="little">
+					BBa_E1010 will be transformed into MG1655 by electro-transformation to make sure.</span>
+					</br>
 				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 791: / Line 884: @@
 			<section id="18">
 				<div class="date">July 18<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">PCR amplification of parts</div>
 				<div id="content_note" >
-					<span id="little">No Special Event!</span>
-					</br>
+					<span>Parts:</br></br>
+					<ul style="list-style-type:square">
+					<li>BBa_E0040(720bp) with primer GFP-F and Plasmid1-R: 740bp</li>
+					<li>BBa_E1010(681bp) with primer Plasmid1-F and mRFP-R: 701bp</li>
+					</ul></br>
+					Template: distributed parts 1uL</span>
+					</br></br>
+					<b>Results</b></br></br>
+					<span id="little">Gel electrophoresis</span></br></br>
+					<div align="center"><img id="figure" alt="2_0718Fig1" src="https://static.igem.org/mediawiki/2012/3/32/KAIST_2nd_07182012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br></br>
+					<span id="little">PCR purification:</br></br>
+					<ul style="list-style-type:square">
+					<li>BBa_E0040: 48.2ng/uL (purity 1.88)</li>
+					<li>BBa_E1010: 104.3ng/uL (purity 1.88)</li>
+					</ul>
+					</span>
+					</br></br>
+					<b>Discussions</b></br></br>
+					<span id="little">The bands of both parts showed right size and yield is considerable. </span></br></br>
 				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 			</section>
 			<section id="19">
 				<div class="date">July 19<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">BBa_E0040 transformants plasmid DNA mini-prep & enzyme cut check</div>
 				<div id="content_note" >
-					<span id="little">No Special Event!</span>
-					</br>
+					<span>
+					Strain: TOP10 - BBa_E0040</br></br>
+					BBa_E0040 (2799bp): GFP (720bp) cloned into pSB1A2 (2079bp)</br></br>
+					</span>
+					<span id="little">
+					Restriction enzyme digestion conditions</br>
+					</span></br>
+					<div align="center"><img id="figure" alt="2_0719Fig1" src="https://static.igem.org/mediawiki/2012/5/5c/KAIST_2nd_07192012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br></br>
+					<span id="little">
+					Incubated in 37℃ for 1hr
+					</span></br>
+					</br></br>
+					<b>Results</b></br></br>
+					<span>DNA Concentration measurement:</br></br>
+						<ul style="list-style-type:square">
+							<li>Colony #1: 161.3ng/uL (purity 1.74)</li>
+							<li>Colony #2: 140.5ng/uL (purity 1.51)</li>
+							<li>Colony #3: 250.6ng/uL (purity 1.83)</li>
+						</ul>
+						</br>
+					</span>
+					<span>Digested DNA gel electrophoresis</span></br></br>
+					<div align="center"><img id="figure" alt="2_0719Fig2" src="https://static.igem.org/mediawiki/2012/8/80/KAIST_2nd_07192012_Fig2.png"></img></div>
+					<div style="clear:both;"></div>
+					</br></br>
+					<b>Discussions</b></br></br>
+					<span id="little">The bands of three parts showed size little bit larger than desired size. This might be due to additional bases, prefix and suffix, attached during partregistry assembly process. Also, yield is considerable.</span>
+					</br>
 				</div>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
+				<div class="note-title">BBa_E1010 electroporation into MG1655</div>
+				<div id="content_note" >
+					<span>
+					Part: BBa_E1010 (mRFP); <i>KanR</i></br>
+					Strain: MG1655</br>
+					</span>
+					</br>
+				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 811: / Line 985: @@
 			<section id="20">
 				<div class="date">July 20<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">MG1655-BBa_E1010 colony inoculation</div>
 				<div id="content_note" >
-					<span id="little">No Special Event!</span>
-					</br>
-				</div>
+					<span id="little">Three colonies were formed. All of them were inoculated in 3mL of LB with 1% of kanamycin.</span>
+					</br>
+				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 821: / Line 999: @@
 			<section id="21">
 				<div class="date">July 21<sup>st</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">BBa_E1010 transformants plasmid DNA mini-prep & enzyme cut check</div>
 				<div id="content_note" >
-					<span id="little">No Special Event!</span>
-					</br>
+					<span>
+					Strain: MG1655</br>
+					BBa_E1010 (5107bp): mRFP (681bp) cloned into pSB2K3 (4426bp)
+					</span>
+					</br></br>
+					<span id="little">Restriction enzyme digestion conditions</span></br></br>
+					<div align="center"><img id="figure" alt="2_0721Fig1" src="https://static.igem.org/mediawiki/2012/5/57/KAIST_2nd_07212012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+					<span id="little">Incubated in 37℃ for 1hr</span></br></br>
+					<b>Results</b></br></br>
+					<span id="little">No band appeared</span>
+					</br>
 				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 831: / Line 1,029: @@
 			<section id="22">
 				<div class="date">July 22<sup>nd</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">pre-culture of MG 1655 cell transformed with <i>Moth 1202</i> gene inserted pTrcHis2A and pBAD/mycHisC vector. </div>
@@ Line 836: / Line 1,036: @@
 					<span id="little">We did pre-culture of both pTrcHis2A <i>1202</i> and pBAD/mycHisC transformed cell into 5ml LB and incubated on 37℃ for 16 hours. </span>
+					</br>
+				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">BBa_E1010 transformants plasmid DNA mini-prep</div>
+				<div id="content_note" >
+					<b>Results</b></br></br>
+					<span id="little">Colony #1: 26.2ng/uL (purity 2.47)</span>
 					</br></br>
+					<b>Discussions</b></br></br>
+					<span id="little">The pSB2K3 is high copy plasmid only if induced by IPTG. Without induction, it is low copy vector. For higher yield, we induce cells with IPTG tomorrow.</span>
+				</div>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
+				<div class="note-title">Primer design for Cre, FLP cloning</div>
+				<div id="content_note" >
+					<div align="center"><img id="figure" alt="2_0722Fig1" src="https://static.igem.org/mediawiki/2012/6/68/KAIST_2nd_07222012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+					<span id="little">Restriction site is suit for both pTrcHis2a and pBADmychisC vector MCS.</span>
+					</br>
 				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 845: / Line 1,075: @@
 			<section id="23">
 				<div class="date">July 23<sup>rd</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title"><i>Moth 0109</i> vector double cut check</div>
@@ Line 869: / Line 1,101: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">Induction of <i>Moth_1202</i> gene (sampling) and running SDS-PAGE gel. </div>
@@ Line 882: / Line 1,114: @@
 			</section>
 			<section id="24">
 				<div class="date">July 24<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Expression check of <i>Moth_1202</i> on pBAD vector (Result)</div>
@@ Line 911: / Line 1,146: @@
 				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">Cre and Flp recombinase gene amplification and purification</div>
+				<div id="content_note" >
+					<span id="little">Cre recombinase (1032bp) and Flp recombinase (1292bp) were amplified from BBa_J61047 and pCP20 respectively using HotStarTaq DNA polymerase. Amplified genes were purified by PCR purification kit.</span>
+					</br></br>
+					<b>Results</b></br></br>
+					<div align="center"><img id="figure" alt="2_0724Fig1" src="https://static.igem.org/mediawiki/2012/9/95/KAIST_2nd_07242012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+					<span id="little">Cre: 175.6 ng/uL (purity: 1.89)</br></span>
+					<span id="little">Flp: 105.3 ng/uL (purity: 1.88)</br></span>
+					</br>
+				</div>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
+				<div class="note-title">Synthesized gene transformation</div>
+				<div id="content_note" >
+					<span id="little">Synthetic constructs of pPoC insert and pPocpi insert were arrived. They were TA cloned into pGEM-B1 vector. We chemically transformed the vectors to DH5α as recommended from BIONEER.</span>
+					</br></br>
+				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 919: / Line 1,186: @@
 			<section id="25">
 			<div class="date">July 25<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Sequencing result of <i>Moth_0109</i></div>
@@ Line 940: / Line 1,208: @@
 				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">Colony PCR</div>
+				<div id="content_note" >
+					<span id="little">Colony PCR of colonies from BBa_E0040 (720bp) and BBa_E1010 (681bp) master plates of July 17<sup>th</sup>. </span>
+					</br></br>
+					<b>Results</b></br></br>
+					<div align="center"><img id="figure" alt="2_0725Fig1" src="https://static.igem.org/mediawiki/2012/b/b2/KAIST_2nd_07252012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+				</div>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
+				<div class="note-title">Inoculation of cells having P1-pGEM and PI-pGEM</div>
+				<div id="content_note" >
+					<span id="little">Inoculated the transformed cells into 3mL of LB broth with 1% of ampicillin.</span>
+					</br>
+				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 947: / Line 1,239: @@
 			<section id="26">
 				<div class="date">July 26<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Vector mini-prep for all genes except <i>0109</i> and FDH.</div>
@@ Line 959: / Line 1,253: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">colony PCR of <i>Moth_0109</i> gene</div>
@@ Line 973: / Line 1,267: @@
 				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
-				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
+				<div class="note-title">vector preparation and transformation check</div>
+				<div id="content_note" >
+					<span id="little">MG1655 strains that carrying pTrcHis2A (4.4kb) and pBAD/Myc-HisC (4.1kb) were inoculated. We extracted plasmid DNA from the cells.</br></br>
+					  P1-pGEM and PI-pGEM plasmids were also extracted.</br></br>
+					  All the plasmids were checked with restriction enzyme cut.</br>
+					<ul style="list-style-type:square">
+						<li>pTrcHis2A and pBAD/Myc-HisC with EcoRI</li>
+						<li>P1-pGEM and PI-pGEM with HindIII</li>
+					</ul>
+					</span>
+					</br></br>
+					<b>Results</b></br></br>
+					<div align="center"><img id="figure" alt="2_0726Fig1" src="https://static.igem.org/mediawiki/2012/1/1c/KAIST_2nd_07262012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+				</div>
+				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
 			</section>
 			<section id="27">
 				<div class="date">July 27<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Primer design for Gibson assembly</div>
@@ Line 998: / Line 1,315: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">PCR amplification of fragments for Gibson assembly</div>
@@ Line 1,025: / Line 1,342: @@
 					</br>
 				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">pPoC insert and pPocpi insert construction: Trial1</div>
+				<div id="content_note" >
+					<span id="little">pPoc insert consists of BBa_E1010, P1 and BBa_E0040 and pPoC insert consists of BBa_E1010, PI and BBa_E0040 were constructed using Overlapping Extension(OE) PCR.</br></br>Template preparation</br></br></span>
+					<div align="center"><img id="figure" alt="2_0727Fig1" src="https://static.igem.org/mediawiki/2012/6/64/KAIST_2nd_07272012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					<span>OE PCR</span></br>
+					<div align="center"><img id="figure" alt="2_0727Fig2" src="https://static.igem.org/mediawiki/2012/1/1d/KAIST_2nd_07272012_Fig2.png"></img></div>
+					<div style="clear:both;"></div></br>
+					<span>We mixed each template with equimolar amount.</span></br>
+					</br>
+					<b>Results</b></br></br>
+					<div align="center"><img id="figure" alt="2_0727Fig3" src="https://static.igem.org/mediawiki/2012/6/67/KAIST_2nd_07272012_Fig3.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+					<span id="little">Failed to construct the insert sequences of pPoC and pPoCpi.</span></br>
+					</br>
+					<b>Discussions</b></br></br>
+					<span id="little">We missed adding pfu-X polymerase when amplifying templates and OE PCR. We think that is the reason why we failed to construct inserts.</span>
+					</br>
+				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 1,033: / Line 1,381: @@
 			<section id="28">
 				<div class="date">July 28<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">pre-culture of pTrcHis2A-<i>Moth0109</i> vector inserted MG1655 cell. </div>
@@ Line 1,041: / Line 1,391: @@
 				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">pPoC insert and pPocpi insert construction: Trial2</div>
+				<div id="content_note" >
+					<span id="little">Template preparation</span>
+					</br></br>
+					<div align="center"><img id="figure" alt="2_0728Fig1" src="https://static.igem.org/mediawiki/2012/9/93/KAIST_2nd_07282012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+					<div align='center'>
+					<span>pPoC & pPoCpi promoter segment(214bp) </span>
+					</div>
+					</br></br>
+					<span id="little">OE PCR</span>
+					</br></br>
+					<div align="center"><img id="figure" alt="2_0728Fig2" src="https://static.igem.org/mediawiki/2012/1/13/KAIST_2nd_07282012_Fig2.png"></img></div>
+					<div style="clear:both;"></div>
+					</br>
+					<div align='center'>
+					<span>→ Gel extracted</span>
+					</div>
+					</br></br>
+					<b>Discussions</b></br></br>
+					<span id="little">When performing OE PCR, adding forward and reverse primers of full fragment at the first time make the yield higher than without adding primers.
+					</span>
+					</br>
+				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 1,048: / Line 1,429: @@
 			<section id="29">
 				<div class="date">July 29<sup>th</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">PCR amplification of pBAD - <i>Moth_1191</i> & <i>1199</i></div>
@@ Line 1,070: / Line 1,453: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">pre-culture of cell for vector miniprep - <i>Moth_1191</i> & <i>1199</i></div>
@@ Line 1,081: / Line 1,464: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">Induction of <i>Moth_0109</i> gene (sampling) and running SDS-PAGE gel. </div>
@@ Line 1,090: / Line 1,473: @@
 					</br></br>
 				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">pPoC, pPoCpi Cloning</div>
+				<div id="content_note" >
+					<span id="little">Cloning Conditions – iGEM recommended protocol</span></br></br>
+					<span>
+						<ol>
+						<li>Restriction enzyme digestion</li></br>
+						</ol>
+						<ul style="padding:0px 0px 0px 60px;">
+						<li>Enzyme pre-mix – 37℃ 1hr, 80℃ 20min</li></br>
+						<div align="center"><img id="figure" alt="2_0729Fig1" src="https://static.igem.org/mediawiki/2012/0/03/KAIST_2nd_07292012_Fig1.png"></img></div>
+						<div style="clear:both;"></div>
+						</br>
+						<li>Digestion mixture – 37℃ 1hr, 80℃ 20min</li></br>
+						<div align="center"><img id="figure" alt="2_0729Fig2" src="https://static.igem.org/mediawiki/2012/d/df/KAIST_2nd_07292012_Fig2.png"></img></div>
+						<div style="clear:both;"></div>
+						</br>
+						</ul>
+						<ol start='2'>
+						<li>Ligation - 16℃ 1hr, 80℃ 20min</li></br>
+						</ol>
+						<div align="center"><img id="figure" alt="2_0729Fig3" src="https://static.igem.org/mediawiki/2012/c/ca/KAIST_2nd_07292012_Fig3.png"></img></div>
+						<div style="clear:both;"></div>
+						</br>
+						</br>
+					</span>
+					<span id="little">Transformed into DH5α chemically competent cell.</span>
+					</br>
+				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
@@ Line 1,096: / Line 1,513: @@
 			<section id="30">
 				<div class="date">July 30<sup>th</sup> 2012</div></br>
+ 				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">Expression optimization of <i>Moth_0109</i> gene on pTrcHis2A (Result)</div>
 				<div id="content_note" >
-				<!--맞는 지 모르겠음..ㅠㅠ도대체 뭘 내린건지..	-->
 					<span id="little">For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94 kDa protein, formate tetrahydrofolate ligase. </span>
 					</br></br>
@@ Line 1,110: / Line 1,528: @@
 					</br>
 					<b>Discussion</b></br></br>
-					<!--Band가 정확하지가 않았네….-->
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene (3rd and 4th trial)</div>
@@ Line 1,137: / Line 1,553: @@
 					<span id="little">We thought that there might be some problem with vector preparation, so we tried mini-prep once more, and we got no band. Also, we tried colony PCR of <i>1191</i> and <i>1199</i> gene with Gibson primers, which has failed. </span>
+					</br>
+				</div>
+				</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+				<div class="note-title">Ligation product plating</div>
+				<div id="content_note" >
+					<b>Results</b></br></br>
+					<span id="little">No colony appeared</span>
+					</br></br>
+					<b>Discussions</b></br></br>
+					<span id="little">Maybe iGEM recommended protocol is inappropriate or we have to increase our insert DNA concentration.</span>
+					</br>
+				</div>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
+				<div class="note-title">Primer design for pPoC and pPoCpi</div>
+				<div id="content_note" >
+					<span id="little">We decided not to use linearized plasmid backbone. Thus we designed primers which have EcoRI and PstI as its restriction site.</span>
+					</br></br>
+					<div align="center"><img id="figure" alt="2_0730Fig1" src="https://static.igem.org/mediawiki/2012/a/aa/KAIST_2nd_07302012_Fig1.png"></img></div>
+					<div style="clear:both;"></div>
 					</br>
 				</div>
@@ Line 1,146: / Line 1,593: @@
 			<section id="31">
 				<div class="date">July 31<sup>st</sup> 2012</div></br>
+				<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 				<div class="note-title">PCR amplification of <i>Moth_1191</i>, <i>1199</i> gene (5th trial)</div>
@@ Line 1,166: / Line 1,615: @@
 				</div>
-				</br><hr></br>
+				</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 				<div class="note-title">pre-culture of pBAD/mycHisC-<i>Moth0109</i> vector inserted MG1655 cell. </div>
@@ Line 1,176: / Line 1,625: @@
 				</div>
 				<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>

Team:KAIST Korea/Notebook Labnote/2012 7

From 2012.igem.org

Latest revision as of 01:00, 27 October 2012

Labnote