Team:Tokyo-Nokogen/notebook

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Latest revision as of 00:15, 22 October 2009

★ 07/13/09 to 07/17/09---start of project

Discuss our team project and set goals

Collect and search journals and information related to our project

★ 07/20/09 to 07/24/09---start of project

Research the Registry for Biobrick

★ 07/27/09 to 07/31/09

Laboratory event (summer vacation)

★ 08/03/09 to 08/07/09---preparation for expariment

Prepare for experiment (make LB medium, several antibiotic LB plates and DH5a competent cell)

Transform and do miniprep plasmids contain standard parts.

(BBa_J23100, BBa_J23110, BBa_J23112, BBa_B0034, BBa_B1105, BBa_E0040 and BBa_E1010)

★08/10/09 to 08/14/09 ---preparation for experiment

Transform and miniprep standard parts (RBS, BBa_I15008, BBa_I15009, BBa_S03417, BBa_R0083,

GFP, Terminator, BBa_I714075, crRBS, BBa_K145001, BBa_I719005, RFP, BBa_K113009 and

BBa_K124003. Search DNA sequence of anitigen43 and find that there is 6 PstⅠ restriction sites.

★ 08/17/09 to 08/21/09 ---parts construction

[counter group】

Cut plasmids by restriction enzymes and pick up RBS and taRNA-Term. Ligate these parts and DH5a

was transformed with sample. Do same things with following parts.

	Part 1	Part 2
[c1]	RBS	taRNA-Term
[c2]	crRBS	T7RNA polymerase
[c3]	[c2]	termintor

This chart suggests that we pick up part 1 and part 2 materials, ligate them, transform DH5a with them,

inoculate that cell and do miniprep after cultivation.

【red light receptor】

Construct parts

	part 1	part 2
[r1]	RBS	Ho1
[r2]	RBS	PcyA

【aggregation and lysis】

Order primers which designed to amplify antigen43.

Construct parts.

	part 1	part 2
[a1]	Lysis Cassette	terminator
[a2]	Lysis Cassette S105	Terminator

★ 08/24/09 to 08/28/09 ---parts construction

【counter】

Construct parts

	part 1	part 2
[c4]	T7 promoter	crRBS
[c5]	[c3]	[c4]
[c6]	GFP	Terminator

【red light receptor】

Construct parts

	part 1	part 2
[r3]	[r1]	[r2]
[r4]	[r3]	RBS+cph8+terminator
[r5]	OmpR(+)promoter	RBS
[r6]	GFP	terminator

【aggregation and lysis】

Extract genome contains antigen43 from E.coli K-12.

Amplify antigen43 by PCR using primers we made.

Construct parts

	part 1	part 2
[a3]	pBad/araC	RBS
[a4]	[a3]	[a1]
[a5]	[a3]	[a2]

★ 08/31/09 to 09/04/09 -----parts construction

【counter】

Construct parts.

	part 1	part 2
[c7]	[c5]	[c6]
[c8]	T7 RNA polymerase	terminator
[c9]	[c4]	[c8]

Make shortage plates and LB medium.

【red light receptor】

	part 1	part 2
[r7]	[r5]	[r6]

【aggregation and lysis】

Check sequence of [a1] and [a2], but cannot read it.

Gel electrophoresis of antigen43 and expression vector pTrc99-NdeⅠ cut by NdeⅠ,

but cannot find appropriate bands.

★ 09/07/09 to 09/11/09

【counter】

	part 1	part 2
[c10]	[c9]	[c4]
[c11]	RFP	Terminator

【green light receptor】

	part 1	part 2
[g1]	RBS	GlrN

【aggregation and lysis】

Gel electrophoresis of antigen43 cut by NdeⅠand SpeⅠ.

Gel electrophoresis of expression vector pTrc99-NdeⅠ cut by NdeⅠand XbaⅠ

Do geneclean both of them, ligate them, transform DH5a with ligated sample and inoculate it.

Check sequence of [a1] and [a2] again, but cannot read it.

★ 09/14/09 to 09/18/09

【counter】

	part 1	part 2
[c12]	[c10]	[c11]

Sequence analysis.

【green light receptor】

	part 1	part 2
[g1]	[g1]	terminator

Sequence analysis

【aggregation and lysis】

Pick up some single colonies from inoculated plate made last week and do direct colony PCR. After PCR,

we check presence of insert DNA in plasmid by electrophoresis but cannot find.

Order primers which break 6 PstⅠsites not to change amino acid sequence.

Try reconstruction of [a1] to [a3].

★ 09/21/09 to 09/25/09

【counter】

From results of sequence analysis, we find we couldn’t construct all of the above counter parts.

And start reconstruction of counter parts ([c1], [c2] [c4] and following parts.)

	part 1	part 2
[c13]	high promoter	cr-RBS
[c14]	[c1]	[c13]

【green light receptor】

	part 1	part 2
[g3]	[r3]	[g2]

Sequence analysis

【aggregation and lysis】

Gel electrophoresis of antigen43 cut by NdeⅠand SpeⅠ.

Gel electrophoresis of expression vector pTrc99-NdeⅠ cut by NdeⅠand XbaⅠ

Do geneclean both of them, ligate them, transform DH5a with ligated sample and inoculate it.

Try reconstruction of [a4] and [a5].

★ 09/21/09 to 09/25/09

【counter】

Reconstruction of parts ([c3], [c6], [c12] and follwing part)

	part 1	part 2
[c15]	[c14]	[c12]
[c16]	[c4]	[c6]

Check sequence of reconstruction parts.

【light receptor】

Test red light receptor.

【aggregation and lysis】

Pick up some single colonies from inoculated plate made last week and do direct colony PCR.

After PCR, we check presence of insert DNA in plasmid by electrophoresis but cannot find.

Pick up all colonies and cultivate them and do induction in order to watch out occurrence of aggregation.

But we cannot see aggregation.

★ 09/28/09 to 10/02/09

【counter】

Reconstruction of parts ([c5] and following parts)

	part 1	part 2
[c17]	pBad/araC	[c15]
[c18]	[c3]	[c16]

Sequence analysis

【light receptor】

Test green light receptor.

【aggregation and lysis】

Try TA cloning (ligation of antigen43 with pGEMT vector).

Transform DH5a with ligated sample, inoculate it, cultivate it and do miniprep.

Try mutation 1st and 2nd PstⅠ sites. After mutagenesis, transform DH5a with ligated sample, inoculate it.

But there is no colony on plate.

★ 10/5/09 to 10/09/09

【counter】

	part 1	part 2
[c19]	High promoter	[c18]

Sequence analysis

【aggregation and lysis】

Check sequence of [a1] and [a2] again, but cannot read full length sequence.

→ Something wrong with Biobrick parts.

★ 10/12/09 to 10/16/09

Start testing all parts.

Prepare for sending parts to MIT

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