Digestion:transformation of parts listed as below.

•lactose promoter（ampicilin）

•double terminator（ampicilin）

•4-17M:BBa_K325909(chloramphenicol)

•1-12M:BBa_E0240（ampicilin）

•2-17F:BBa_120260(kanamycin)

After transformation, we put E.coli in the plate which is containing the each antibiotic.

We failed to conduct transformation of 4-17M,1-12M,and2-17F ,but transformation of lactose promoter and double terminator worked out.

Digestion:retry of transformation

We could not work out transformation of 4-17M,1-12M,and2-17F. This was why we tried again.

However, these experiments were failed.

Digestion:liquid culture of lactose promoter and double terminator

Picked colonies by using tips ,then put in the LB medium（LB　3ml ampicilin 3μl）

Digestion:Mini prep of lactose promoter and double terminator.

This is results of the experiment.

•lactose promoter 74.3μl/ml

•double terminator 6.7μl/ml

lactose promoter expressed GFD,so we judged this part was not correct one.

Digestion:Transformation of parts listed as below

4-17M（luciferage）

1-12M（GFP-dT）

2-17F（middle copy vector:iGEM 2009kit）

these transformation were failed.

Digestion:Transformation of these parts as below

4-17M:BBa_k325909（luciferage）

1-12M:BBa_E0249（GFP-dT）

2-17F:BBa_I20260（middle copy vector:iGEM 2010kit）

these transformation were failed.

Digestion:Transformation

•1-5E（pSB3K3）

Digestion:DNeasy of these parts described as below

•JCM4616 16.9μg/ml 1.48 260/280

•JCM5070 7.7μg/ml 1.40 260/280

Digestion:liquid culture

•double terminator

•1-5E

Digestion:Restriction enzyme digestion

•double terminator（77.4μg/ml）

result

this experiment was failed.

the reason of this failure was

Digestion:Miniprep

•1-5E　7.1μg/ml 1.53 260/280

Digestion:Transformation of a part described as below.

•1-6G:BBa_R011（pSB1A2）

Digestion:DNeasy of these parts described as below.

•JCM4616　4.4μg/ml 1.22 260/280 0.28 260/230

•JCM5070 3.2μg/ml 1.38 260/280 0.25 260/230

Digestion:electrophoresis

•double terminator（77.4μg/ml）

•1-5E（7.1μg/ml）

results

Digestion: Transformation of 1-5E
Digestion: liquid culture of JCM5070 and JCM4616

Digestion（nobeyama izumi komatsu）:DNeasy of JCM5070 and JCM4616
these results were described as below
JCM5070-1　5.2μg/ml
JCM5070-2 4.9μg/ml
JCM5070-3 4.4.μg/ml
JCM4616-1 10.7μg/ml
JCM4616-2 10.0μg/ml
JCM4616-3 16.2μg/ml

Digestion:liquid culture of 1-6E,JCM5070,and JCM4616

Digestion: Mini prep of 1-6E-1,1-6E-2,and1-6E-3.

RESULTS

1-6G-1 there were no plasmid.

1-6G-2 there were no plasmid.

1-6G-3 132.3μg/ml 1.77 260/280 2.16 260/230

Digestion:Mini prep of 1-6E,σ54-5,and σ54-10

these results were described as below

1-6E-1 failure

1-6E-2 failure

1-6E-3 failure

σ-54-5 26.4μg/ml 1.70μg/ml 260/280 1.60μg/ml 260/230

σ-54-10 failure

Digestion:DNeasy of JCM5070 and JCM 4616.

JCM5070W1 　7.1μg/ml

JCM5070W2 　13.6μg/ml

JCM5070G1　　8.1μg/ml

JCM5070G2　　4.8μg/ml

JCM4616W1　　64.9μg/ml

JCM4616W2　　7.5μg/ml

JCM4616G1　　10.1μg/ml

JCM4616G2　　 8.1μg/ml

Digestion;retry of liquid culture of 1-6G-1 and 1-6G-2

Digestion: Miniprep of 1-6G which was cultured on September 5

these results were described as below.

1-6G-1 139.1μg/ml 1.63 260/280 2.10 260/230

1-6G-2 130.3μg/ml 1.75 260/280 2.19 260/230

Digestion:ethanol precipitation of JCM5070W2（1st September）and JCM4616-3（31th August）.

these results were described as below.

•JCM5070W2 37.2μg/ml 1.20 260/280 0.65 260/230

•JCM4616-3 53.0μg/ml 1.13 260/280 0.58 260/230

Digestion(Mori)

PCR amplification of SAM-P20 and ChiA

We performed colony direct PCRs from a S. albogriseolus colony and a S. avermitilis colony.

Reaction mixture

Component	Volume(μl)
2x Buffer	25
2mM dNTPs	10
Primer 1	1.5
Primer 2	1.5
Template	X
KOD FX	1
ddH2O	up to 50

PCR condition

Predenature	94C	2m
Denature	98C	10s	30cycles
Annealing	56C	30s
Extension	68C	1m30s

We prepared two kinds of templates:

1. Picked a colony, suspended in 50ul of water and incubated 1min at 95 degree. Added 1ul to the reaction mixture.
2. Picked a colony and dipped in the reaction mixture.

Gel electrophoresis indicated that the PCR amplifications were successful for a sample, ChiA, but it was a faint band. We decided to retry direct PCR of SAM-P20 and PCR of ChiA.

Digestion(Mori)

Retry of PCR amplification of SAM-P20 and ChiA.

We amplified SAM-P20 by colony direct PCR and ChiA by PCR using the PCR product that performed yesterday.

Reaction mixture: The same components and volume as before.

PCR condition: The same PCR condition as before.

PCR for SAM-P20

We prepared two kinds of templates:

1. Picked a colony, suspended in 50ul of TE buffer (pH8.0) and incubated 1min at 95 degrees. Added 5ul to the reaction mixture.
2. Picked a colony and dipped in the reaction mixture.

PCR for ChiA

1μl of PCR product was added to the reaction mixture as template.

Gel electrophoresis indicated that the PCR amplifications were successful for all samples. However, an unexpected faint band, about 1000bp, was also observed in the sample of ChiA.

PCR purification of SAM-P20 and gel extraction of ChiA.

SAM-P20: 43.9 ng/μl

ChiA: 30.0 ng/μl

Restriction enzyme digestion

We performed restriction digestions for:

1. SAM-P20 with EcoRI and SpeI
2. ChiA with EcoRI and SpeI

Incubated overnight at 37 degrees.

Digestion (Mori)

Purification of digested products

SAM-P20 : 10.2 ng/μl

ChiA : 22.5 ng/μl

Ligation

Name	Vector	Insert
1	pSB1C3	SAM-P20
2	pSB1C3	ChiA
3	BBa_B0015	SAM-P20
4	BBa_B0015	ChiA

Incubated overnight at 16 degrees.

Digestion (Mori)

Transformation

Name	Vector	Insert	Growth
1	pSB1C3	SAM-P20	No
2	pSB1C3	ChiA	Yes
3	BBa_B0015	SAM-P20	Yes
4	BBa_B0015	ChiA	Yes

Digestion (Mori, Nobeyama)

Colony PCR Performed colony PCR to check if ligation and transformation were successful. Gel electrophoresis shown that the ligation and transformation of pSB1C3 with ChiA was successful.

Digestion (Mori)

Measurement assay for chitinase A1.

We performed standard measurement and trial of sample measurement.