Team:Todai-Tokyo/Notebook/isoleucine

 the notebook  p { font-size : 10pt } pz{ font-size : 30pt; color : red} .aaa { font-size : 20pt } @charset "UTF-8"; body { background:black; } div#top_plan{ background:#B22222; color:#ffffe0; margin 0 0 0 0 width:900px; height:200px; } div#exp{ background:#696969; color:#C0C0C0; width:700px; height:200px; } div#exp day{ background:#006400; color:white; width 70px; height 70px; margin: 10 0 10 0; float:left; } = Plan = Aim: Create bacteria that produce a noxious odour when induced to do so Methods:
 * 1) Clone the yqiT gene from Bacillus subtilis into a biobrick vector.
 * 2) Make constructs that express this gene constitutively and under the control of an inducible promoter.

=June= First, we haven't decided what promoter to use. While looking for a good promoter, we were cloning YqiT and useful parts.

6/5
Constructs to be created:


 * 1) yqiT
 * 2) ptetR-RBS-yqiT-dterm
 * 3) pAraC-RBS-yqiT-dterm
 * 4) pLacI-RBS-yqiT-dterm

Obtaining DNA:

Resuspended DNA in the following wells with 10ul water:

Plate 1 1D AraC regulated promoter Plate 1 12E LacI regulated promoter

Plate 1 1H Strong RBS

Plate 1 13B TetR repressed POPS generator

Plate 2 24C Double terminator

Transformed 1ul of each of the above into DH5a competent cells:

Transformation
 * Mix 1ul of DNA with 100ul of competent cells on ice.
 * Leave on ice for 30 minutes.
 * Heat shock at 42°Cfor 45 seconds.
 * Leave on ice for 2 minutes.
 * Add 500ul of LB and incubate at 37°C for 1 hour.
 * Plate on LB-ampicillin plates.

6/6
No colonies grew from the 5 transformations of 6/5.

6/7
Creating a biobrick part out of yqiT

Strategy: PCR out the yqiT gene from the Bacillus subtilis genome using primers to attach the biobrick preffix/suffix and clone this into a biobrick vector. The vector utilized is the GFP generator which houses a sufficient-sized insert:

Plate 1 16E (from 2007 iGEM distribution provided by Chiba University)<BR> GFP generator

Overview:<BR>
 * 1) PCR yqiT gene from genome
 * 2) purify DNA from the PCR product
 * 3) digest GFP generator and purified PCR product by XbaI/PstI
 * 4) gel-purify the digested vector (GFP generator without the insert i.e. empty) and the yqiT gene
 * 5) ligate the above 2 fragments of DNA together
 * 6) transform into E. coli and select for ampicillin resistance
 * 7) check for transformation success using colony PCR by yqiT primers

PCR of yqiT gene<Br> The Bacillus subtilis genome was provided by Bacillus subtilis 168

The following primers were used to amplify an approx. 1500bp fragment from the Bacillus subtilis genome.

yqiT EX: ccggaattctctagaatggaactttttaaatatatggaacgatacg(bold text is a sequence binding yqiT gene)<BR> yqiT SP: ctgcagcggccgctactagtattagcgacgacttaaaatatgttgg(bold text is a sequence binding yqiT gene)

PCR protocol<BR> The following was mixed in a PCR tube:<Br> 1ul 20uM yqiT EX primer<Br> 1ul 20uM yqiT SP primer <Br> 2ul 10X Pfu Ultra buffer<Br> 1.6ul dNTP mix<Br> 0.15ul Bacillus subtilis genome<Br> 0.5ul Pfu Ultra enzyme<Br> 13.85ul MilliQ<Br>

Performed PCR using the following program:

1. 95°C 2 minutes<Br> 2. 95°C 30 seconds<Br> 3. 50°C 30 seconds<Br> 4. 72°C 40 seconds<Br> 5. Repeat 2-4 23 times<Br> 6. 25°C forever<Br>

Purified PCR product using the Promega PCR purification kit.

Ran on gel to visualize bands:



PCR unsuccessful : will digest the GFP generator but cannot perform the ligation without the PCR product.

<BR><BR> Digestion<BR> Digested purified PCR product and the GFP generator both with XbaI/PstI:

yqiT<Br> 3ul PCR product<Br> 1ul High buffer<Br> 0.5ul XbaI<Br> 0.5ul PstI<Br> 5ul MilliQ<Br>

GFP generator<Br> 6ul DNA<Br> 2ul High buffer<Br> 1ul XbaI<Br> 1ul PstI<Br> 10ul MilliQ<Br>

Incubated mixtures at 37°C for 1 hour.

Ran on gel to gel purify:<BR> Lanes 1, 12:marker <BR>Lanes 2, 5, 9:GFP <BR>Lanes 3, 6, 10:yqiT<BR>



Cut out the second band from the bottom and dissolved the gel using the Promega gel purification kit to extract DNA from.

This DNA is stored for later usage as the GFP generator X/P vector fragment.

6/14
Creating a biobrick part out of yqiT

Strategy: PCR out the yqiT gene from the Bacillus subtilis genome using primers to attach the biobrick preffix/suffix and clone this into a biobrick vector. The vector utilized is the GFP generator which houses a sufficient-sized insert:

Plate 1 1D (from 2009 iGEM distribution provided by Tokyo University) promoter (lacI regulated)

Overview:

PCR yqiT gene from genome

gel-purify DNA from the PCR product

digest lacI regulated promoter and purified PCR product by XbaI/PstI

gel-purify the digested vector (lacI regulated promoter without the insert i.e. empty) and the yqiT gene

ligate the above 2 fragments of DNA together

transform into E. coli and select for ampicillin resistance

check for transformation success using colony PCR by yqiT primers

PCR of yqiT gene The Bacillus subtilis genome was provided by Bacillus subtilis 168

The following primers were used to amplify an approx. 1095bp fragment from the Bacillus subtilis genome.

yqiT EX: ccggaattctctagaatggaactttttaaatatatggaacgatacg(bold text is a sequence binding yqiT gene)

yqiT SP: ctgcagcggccgctactagtattagcgacgacttaaaatatgttgg(bold text is a sequence binding yqiT gene)

PCR protocol

The following was mixed in a PCR tube:

1ul 20uM yqiT EX primer

1ul 20uM yqiT SP primer

2ul 10X Pfu Ultra buffer

1.6ul dNTP mix

0.15ul Bacillus subtilis genome

0.5ul Pfu Ultra enzyme

13.75ul MilliQ

Performed PCR using the following program:

1. 95°C 2 minutes

2. 95°C 30 seconds

3. 50°C 30 seconds

4. 72°C 40 seconds

5. Repeat 2-4 29 times

6. 25°C forever

Purified PCR product using the Promega PCR purification kit.

Ran on gel to gel purify:

Lanes 1:marker 6ul

Lanes 2:yqiT PCR product 10ul + 10xLoading Dye 2ul<BR> <BR> PCR successful

Ligation

Ligated gel purified yqiT and vector with TaKaRa Solutions 1:

lacI regulated promoter (vector) 2ul

yqiT 8ul

TaKaRa Solutions 1 6ul

Incubated mixtures at 37°C for 10 minutes.

Transformation

6/21
Creating a biobrick part out of yqiT

Strategy: PCR out the yqiT gene from the Bacillus subtilis genome using primers to attach the biobrick preffix/suffix and clone this into a biobrick vector. The vector utilized is the GFP generator which houses a sufficient-sized insert:

Plate 1 1D (from 2009 iGEM distribution provided by Tokyo University)<BR> promoter (lacI regulated)

Overview:<BR>
 * 1) PCR yqiT gene from genome
 * 2) gel-purify DNA from the PCR product
 * 3) digest lacI regulated promoter and purified PCR product by XbaI/PstI
 * 4) gel-purify the digested vector (lacI regulated promoter without the insert i.e. empty) and the yqiT gene
 * 5) ligate the above 2 fragments of DNA together
 * 6) transform into E. coli and select for ampicillin resistance
 * 7) check for transformation success using colony PCR by yqiT primers

PCR of yqiT gene<Br> The Bacillus subtilis genome was provided by Bacillus subtilis 168

The following primers were used to amplify an approx. 1095bp fragment from the Bacillus subtilis genome.

yqiT EX: ccggaattctctagaatggaactttttaaatatatggaacgatacg(bold text is a sequence binding yqiT gene)<BR> yqiT SP: ctgcagcggccgctactagtattagcgacgacttaaaatatgttgg(bold text is a sequence binding yqiT gene)

PCR protocol<BR> The following was mixed in a PCR tube:<Br> 1ul 20uM yqiT EX primer<Br> 1ul 20uM yqiT SP primer <Br> 2ul 10X Pfu Ultra buffer<Br> 1.6ul dNTP mix<Br> 0.15ul Bacillus subtilis genome<Br> 0.5ul Pfu Ultra enzyme<Br> 13.75ul MilliQ<Br>

Performed PCR using the following program:

1. 95°C 2 minutes<Br> 2. 95°C 30 seconds<Br> 3. 50°C 30 seconds<Br> 4. 72°C 40 seconds<Br> 5. Repeat 2-4 29 times<Br> 6. 25°C forever<Br>

Purified PCR product using the Promega PCR purification kit.

Ran on gel to gel purify:<BR> Lanes 6:marker 6ul <BR>Lanes 2, 4:yqiT PCR product 10ul + 10xLoading Dye 2ul<BR>



PCR successful

<BR><BR> Digestion<BR> Digested purified PCR product and the lacI regulated promoter both with XbaI/PstI:

yqiT<Br> 3ul PCR product<Br> 1ul High buffer<Br> 0.5ul XbaI<Br> 0.5ul PstI<Br> 5ul MilliQ<Br>

lacI regulated promoter<Br> 4ul DNA<Br> 4ul High buffer<Br> 2ul XbaI<Br> 2ul PstI<Br> 28ul MilliQ<Br>

Incubated mixtures at 37°C for 1 hour.

Ran on gel to gel purify:<BR> Lanes 3:marker 6ul <BR>Lanes 2, 5:lacI regulated promoter 40ul + 10xLoading Dye 8ul <BR>Lanes 1, 4:yqiT 10ul + 10xLoading Dye 2ul<BR>



Cut out the band and dissolved the gel using the Promega gel purification kit to extract DNA from.

Ligation<BR> Ligated gel purified yqiT and vector with TaKaRa Solutions 1:<BR>

lacI regulated promoter (vector) 0.5ul<BR> yqiT 5.5ul<BR> TaKaRa Solutions 1 4ul<BR>

Incubated mixtures at 37°C for 10 minutes.

Transformation<BR>

=July= We tried to read the sequence of YqiT for the first time.<BR> But the trial　ended to failure....

7/5
TA cloning of YqiT<BR> PCR of YqiT<BR> 0.25ul Takara ExTaq<BR> 5ul 10xExTaq Buffer<BR> 4ul dNTPmix<BR> 0.5ul Bacillus subtilis genome<BR> 0.5ul 5'primer(It is the same sequence as that on 6/21)<BR> 0.5ul 3'primer(It is the same sequence as that on 6/21)<BR> up to 50ul MilliQ

Performed PCR using the following program: 1. 94°C 2 minutes<Br> 2. 94°C 30 seconds<Br> 3. 52°C 80 seconds<Br> 4. 72°C 60 seconds<Br> 5. Repeat 2-4 29 times<Br> 6. 25°C forever<Br>

lane1:marker<BR> lane2~5:YqiT<BR>

<BR> PCR successful!<BR> But we couldn't do ligation because TA cloning kit was not left.<BR> We purified PCR products and put them in -20°C.

7/19
YqiT colony PCR<BR>

put a small amount of single colony into each tube with 5ul MilliQ water<BR> v<BR>
 * <BR>

95°C 5min<BR> v<BR>
 * <BR>

PCR reaction<BR> 1ul 10×buffer <BR> 0.8ul 2.5mMdNTP<BR> 0.08ul Ex-Taq<BR> 0.1ul 5’-primer<BR> 0.1ul 3’-primer<BR> 2.92ul MilliQ water<BR> v<BR>
 * <BR>

added the PCR reaction to each tube.<BR> v<BR>
 * <BR>

Performed PCR using the following program:<BR> 1. 95°C 2min<BR> 2. 95°C 30sec <BR> 3. 52°C 30sec<BR> 4. 72.5°C 80sec<BR> 5. repeat 2-4 29times<BR> 6. 25°C forever<BR>

PCR successful!<BR>

added a small amount of colony number 2 and 3 to each test tube with 4ml LB broth.<BR> cultured them over night.<BR>

7/20
Miniprep of E.coli cells containing YqiT gene with Promega, Wizard Plus SV Miniprep DNA Purification System

7/27
Sequencing YqiT by BIG DYE<BR> 1.8ul 5xB.D.3.1.buffer<BR> 0.4ul B.D.3.1.<BR> 6.3ul MilliQ water<BR> 1ul plasmid(0.15ug/ul)<BR> 0.5ul 5'or3'primer(3.2pmol/ul)<BR> v<BR>
 * <BR>

PCR　Program<BR> 1.96°C 2min<BR> 2.96°C 10sec<BR> 3.55°C 5sec<BR> 4.60°C 3min <BR> 5.go to 2.29times<BR> 6.25°C forever<BR> v<BR>
 * <BR>

add 0.5ul PHOSPHATASE ALKALINE shrimp<BR> v<BR>
 * <BR>

37°C 1hr incubate<BR> v<BR>
 * <BR>

add 1ul 3MNaOAc<BR> v<BR>
 * <BR>

add 25ul EtOH<BR> v<BR>
 * <BR>

20000xg 4°C 10min centrifugation<BR> v<BR>
 * <BR>

put off supernatant<BR> v<BR>
 * <BR>

dry tubes<BR> v<BR>
 * <BR>

add 15ul HiDi<BR> v<BR>
 * <BR>

put them in the sequence machine<BR>

7/30~8/2
Sequencing YqiT by BigDye<BR> unsuccessful<BR>

=August= Sequence reading succeed!<BR> We made YqiT + double terminator.

8/3
sequence YqiT by BigDye<BR> successful<BR>

8/7~9
Digestion<BR> insert YqiT gene into a terminator of iGEM parts<BR> terminator<BR> double terminatorMiniprep ., using Promega kit<BR> plate1 23L<BR>

8/12~14
PCR of YqiT for restriction enzyme fragmentation and infusion<BR> PCR protocol<BR> The following was mixed in a PCR tube:<Br> 0.2ul 100uM yqiT EX primer/yqiT infusion 5'primer<Br> 0.2ul 100uM yqiT SP primer/yqiT infusion 3'primer <Br> 2ul 10X Pfu Ultra buffer<Br> 1.6ul dNTP mix<Br> 0.2ul plasmid containing YqiT gene(that sequence matches YqiT gene of Batillus genome) <Br> 0.5ul Pfu Ultra enzyme<Br> 15.3ul MilliQ<Br>

Performed PCR using the following program:

1. 95°C 2 minutes<Br> 2. 95°C 30 seconds<Br> 3. 55°C 30 seconds<Br> 4. 72.5°C 30 seconds<Br> 5. Repeat 2-4 29 times<Br> 6. 25°C forever<Br> <BR> restriction enzyme fragmentation of double terminator by XbaI<BR> 1ug plasmid<BR> 10ul BSA<BR> 10ul 10xM buffer<BR> 2.5ul XbaI<BR> up to　100ul MilliQ water<BR> 37°C over night<BR> restriction enzyme fragmentation of double terminator by EcoRI<BR> 1ug plasmid<BR> 10ul 10xH buffer<BR> 2.5ul EcoRI<BR> up to　100ul MilliQ water<BR> 37°C over night<BR> restriction enzyme fragmentation of YqiT gene by SpeI<BR> 1ug DNA<BR> 10ul 10xH buffer<BR> 2.5ul SpeI<BR> up to　100ul MilliQ water<BR> 37°C over night<BR> restriction enzyme fragmentation of YqiT gene by EcoRI<BR> 1ug DNA<BR> 10ul 10xH buffer<BR> 2.5ul EcoRI<BR> up to　100ul MilliQ water<BR> 37°C over night<BR>

8/15,16
ligate YqiT and double terminator<BR><BR>

8/19
1 colony PCR of YqiT<BR> Result: successful<BR><BR>

=September= Aim<BR> We decided to use YqiT as a reporter of dioxin!<BR> So, we began to create this construct<BR> 1.-XRE-Gal1 promoter-yqiT-<BR> 2.-Ahr-←Gal1promoter/Gal10 promoter→-Arnt-<BR>


 * PCR of yqiT and insert it in iGEM parts(plate 1-7D:Gal1 promoter)<BR>

Performed PCR using the following program:<BR> 1. 95°C 2 minutes<BR> 2. 95°C 30 seconds<BR> 3. 55°C 30 seconds<BR> 4. 72.5°C 30 seconds<BR> 5. Repeat 2-4 29 times<BR> 6. 25°C forever<BR>


 * cut YqiT off with EcoRI and SpeI<BR>
 * cut P1-7D off with EcoRI and XbaI<BR>
 * ligate the upper two DNA<BR>

->the result conforms with NCBI datebase<BR>
 * read the Sequencing of Gal1 promoter+yqiT<BR>

9/15
1. Restriction enzyme digestion<BR>
 * P1 7L with E<BR>

2. ligation + transformation<BR>
 * P1 7L + yqit<BR>

3. restriction enzyme digestion<BR>
 * P1 7L with S<BR>

4. restriction enzyme digestion<BR>
 * yqit with X and P<BR>
 * P1 7D with S and P<BR>

9/16
1. Colony PCR<BR>
 * P1 7D+yqit<BR>

2. gel extraction (product of restriction enzyme digestion)<BR>
 * P1 7L (E,X)<BR>
 * P1 7D (S,P)<BR>
 * yqit (X,P)<BR>

3. ligation and transformation<BR>
 * yqit + P1 7D<BR>

9/17
1. colony PCR<BR>
 * P1 7D + yqit<BR>

2. restriction enzyme digestion<BR>
 * yqit with X and P<BR>
 * P1 7D with S and P<BR>
 * P1 7L with E and S<BR>

9/19
1. Ligation->transformation
 * P1 7D+yqit

9/20
1.Sequencing
 * yqit->failure

2. colony PCR
 * P1 7D+yqit->success

9/21
1.Miniprep
 * P1 7D+yqit

9/22
1.Sequencing
 * P1 7D+yqit->failure

9/23
1. restriction enzyme digestion<BR> 2. Ligation->transformation
 * P1 7D     with E and X<BR>
 * P1 7L+yqit with E and X<BR>
 * P1 7D+XRE
 * P1 7D+yqit+XRE

9/24
1. Miniprep 2. restriction enzyme digestion<BR>
 * P2 10F
 * P1 7D     with E and X<BR>
 * P1 7L+yqit with E and X<BR>

9/25

 * insert XRE in P1-7D and P1-7D including yqiT using Infusion kit(clonthech)<BR>
 * gel purification of P1-7D and P1-7D including yqiT<BR>

9/26

 * preculture of E.coli cells containing Ahr and Arnt gene for Miniprep <BR>

9/27

 * Miniprep of E.coli cells containing Ahr and Arnt gene with Promega, Wizard Plus SV Miniprep DNA Purification System

Aim<BR> TA cloning of Ahr, Arnt and gal1/10 promoter<BR> template:human Ahr cDNA and human Arnt cDNA<BR> 1,95°C 2min<BR> 2,95°C 30sec<BR> 3,55°C 30sec<BR> 4,72.5°C 1.5min<BR> 5,go°C to 2, 29times<BR> 6,25°C forever<BR> <BR> <BR>
 * PCR of Ahr and Arnt for TA cloning(Ex-taq, Funakoshi)<BR>

9/29

 * ligation of P17D+YqiT and XRE


 * ligation of P17D and XRE

P17D with S and P
 * restriction enzyme digest


 * sequencing of XRE+P17D

9/30

 * miniprep of ARNT and AMR

primer: EX_XRE3_5_5' J630101P7Dseq3'
 * colony PCR of P17D+XRE

YqiT+D.T. with Pst1 and Xba1
 * restriction enzyme digest

primer: EX_XRE3_5_5' J630101P7Dseq3'
 * again, colony PCR of P17D+XRE

=October= We made the plasmid including XRE+Gal1p+YqiT.<BR> Cloning of Ahr, Arnt and gal1/10p were difficult...

10/2

 * colony PCR of YqiT

10/7
template:S.serevisiae genome DNA<BR> 1,95°C 2min<BR> 2,95°C 30sec<BR> 3,55°C 30sec<BR> 4,72.5°C1min<BR> 5,go°C to 2, 29times<BR> 6,25°C forever<BR> <BR>
 * PCR of gal1/10 promoter (Ex-taq, Funakoshi)<BR>

10/9
template:S.serevisiae genome DNA<BR> 1,95°C 2min<BR> 2,95°C 30sec<BR> 3,55°C 30sec<BR> 4,72.5°C1min<BR> 5,go°C to 2, 29times<BR> 6,25°C forever<BR>
 * PCR of gal1/10 promoter (Ex-taq, Funakoshi)<BR>

10/10
ligate PCR product and T bector<BR> -><BR> put each plasmid into different E.coli<BR> -><BR> Miniprepof them by using Promega kit<BR> -><BR> read each sequence in order to check whether there is plasmids including Ahr, Arnt or gal1/10 promoter<BR> <BR> <BR>
 * TA cloning of Ahr, Arnt and gal1/10 promoter<BR>

10/11
Using iGEM parts, create the construct that produces YqiT steadily or reguratory<BR> P2-10F-YqiT-double terminator<BR> P2-16E-YqiT-double terminator<BR> P1-22A-YqiT-double terminator<BR> P1-21B-YqiT-double terminator<BR> <BR> cut YqiT-double terminator off with XbaI and PstI<BR> cut P2-10F, P2-16E, P1-22A and P1-21B off with SpeI and PstI<BR> v<BR> ligate YqiT-double terminator and iGEM part<BR> v<BR> put each plasmid into different E.coli<BR> v<BR> Miniprep of them by using Promega kit<BR> v<BR> read each sequence in order to check whether there is plasmids including the construct<BR> <BR>
 * check YqiT expression<BR>
 * <BR>
 * <BR>
 * <BR>
 * <BR>

10/12

 * Miniprep of P1,10F with Promega Kit

10/13

 * Sequencing of Arnt, Ahr

10/14

 * Colony PCR of Arnt, Aht, and pGal1/10
 * Sequencing Ahr, Arnt, and pGal1
 * preculture of Arnt and Ahr

10/15

 * Sequencing Arnt, Ahr and pGal1/10

10/16

 * Miniprep of Arnt,Ahr and pGal1/10 with Promega Kit

10/18
1. Sequencing<BR> read the following sequences;<BR>
 * P1-23A+yqit<BR>
 * P1-21B+yqit<BR>
 * p1-16E+yqit<BR>
 * XRE+Gal1+yqit<BR>
 * P2 10F<BR>

2. transformation<BR>
 * P2 10F+yqit+double terminator<BR>

3.Miniprep <BR>
 * P2 10F <BR>

4. Restriction enzyme digestion<BR>
 * yqit with X and P <BR>
 * XRE+P1 7D with S and P<BR>

5. Gel extraction(after gel electrophoresis)<BR>
 * yqit (processed with restriction enzymes)<BR>
 * P1 7D processed with restriction enzymes)<BR>

6. Restriction enzyme digestion<BR>
 * yqit with P<BR>

7. Restriction enzyme digestion<BR>
 * P1 7D with S and P<BR>
 * P1 22A with E and P<BR>

8. Sequencing<BR>
 * P1 14L<BR>
 * P1 16E<BR>
 * P1 21B<BR>
 * P1 22A<BR>
 * P2 5F<BR>
 * P2 9H<BR>
 * P2 10F<BR>
 * P2 16E <BR>
 * XRE-Gal1-yqit<BR>

9. Restriction enzyme digestion<BR>
 * P1 7D with S and P<BR>

10. Ligation<BR>
 * XRE and yqit<BR>

11. Transformation<BR>
 * XRE + yqit<BR>

12. restriction enzyme digest<BR>
 * yqit with X and P<BR>
 * P2 10F with S and P<BR>

10/19
1. colony PCR<BR>
 * P1 7D+XRE+yqit+double terminator<BR>

2. restriction enzyme digest<BR>
 * P2 10F with S and P<BR>
 * yqit with X and P<BR>

3. sequencing<BR>
 * P2 10F<BR>

4. Ligation<BR>
 * P2 10F + yqit<BR>

5. transformation<BR>
 * P2 10F + yqit<BR>
 * yqit<BR>

6. restriction enzyme digest<BR>
 * XRE+P1 7D+yqit with E and P<BR>

7. infusion<BR>
 * yqit + PET<BR>

8. colony PCR<BR>
 * P1 7D+XRE+yqit<BR>

9. colony PCR<BR>
 * P2 10F+yqit<BR>

10/20~
1. colony PCR<BR>
 * P2 10F+yqit<BR>

2. Smell Test 2. Smell Test<BR>

'''<BR>
 * Time for shake culture (each of sample) 15 hours<BR>
 * Negative control has plasmid containing “yqiT + double terminator” (Not including promoter)<BR>
 * Cleansed air is cleansed with active carbon filter.<BR>
 * Equipment<BR>
 * Trade name: ODOR CONCENTRATION METER<BR>
 * Model: XP-329<BR>
 * Serial No. 311059<BR>
 * Manuf. NEW COSMOS ELECTRIC CO,LTD.<BR>
 * Setting: Peak hold<BR>
 * the following values indicate intensity of the smell<BR>
 * cleansed air:200<BR>
 * air:515<BR>
 * normal E.coli: 582<BR>
 * E.coli with constitutive promotor+RBS+yqit+double terminator: 686<BR>
 * yqit successfully worked!!<BR>
 * '''Associate Prof. Kawanishi offered us the yeast, whose chromosome contains AhR and ARNT.

Result
We have made E.coli which has "Constitutive promoter (strong) - RBS - yqiT - double terminator" in its plasmid. <BR>and<BR> We will have made yeast which has Ahr and ARNT in its chromosome, and "XRE - Gal1p - yqiT" in its plasmid by 21 October.