Team:XMU-China/Content parts

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Parts

Table 1. The Bio-bricks used in the study

Part	Backbone	Type	Location	Size (bp)		Description
Part	Backbone	Type	Location	part	backbone	Description
BBa_K546000	pSB1C3	Signaling	2013-P1-12D	1964	2070	Lux pL controlled LuxR with lux pR autoinducing LuxI (lva tag)- AHL.
BBa_I763020	pSB1C3	Intermediate RBS-GFP-TT	2013-P3-11H	914	2070	RBS-GFP (+LVA Tag)-Terminators
BBa_F2621	pSB1A2	Signaling	2013-P2-21F	1158	2079	3OC6HSL Receiver Device
BBa_K546001	pSB1C3	Device	2013-P1-12F	2135	2070	AIIA AHL Reporter and Quencher
BBa_J04450	pSB4K5	Reporter	2013-P5-5G	1069	3419	RFP Coding Device
BBa_J04450	pSB3T5	Reporter	2013-P5-7C	1069	3241	RFP Coding Device
BBa_J23022	BBa_J23006	Composite	2013-P5-3H	234	2356	[key3d][TT]

Favorite XMU-China 2013 iGEM Team Parts

W/N	Name	Type	Description	Designer	Length
W	BBa_K1036000	Composite	lux pL controlled luxR with lux pR controlled ndh (LVA-tag) coding for NADH dehydrogenase II	Shengquan Zeng Zhaopeng Cheng	2687
W	BBa_K1036001	Coding	the ndh gene coding for respiratory NADH dehydrogenase II	Shengquan Zeng Zhaopeng Cheng	1366
W	BBa_K1036003	Composite	lux pL controlled luxR with lux pR controlled gfp (LVA-tag)	Xin Guo Xi Xi	4052
W	BBa_K1036006	Composite	lux pL controlled luxR with lux pR controlled sfgfp (with LVA-tag)	Zhaopeng Cheng Xiyu Wu	4046

XMU-China 2013 iGEM Team Parts Sandbox

W/N	Name	Type	Description	Designer	Length
W	BBa_K1036002	Composite	gfp (with LVA-tag) under plux R control	Xin Guo Xi Xi	2866
W	BBa_K1036004	Reporter	sfgfp with LVA-tag	Zhaopeng Cheng Xiyu Wu	753
W	BBa_K1036005	Composite	sfgfp (with LVA-tag) under plux R control	Zhaopeng Cheng Xiyu Wu	2880

Agarose Gel Electrophoresis Confirmation for all Plasmids

pSB1C3-gfp-luxI

This plasmid consists of two parts of target genes: gfp and luxI, and both of them are regulated by quorum sensing promoter. High copy number. (Fig.7-1)

Fig.7-1 AGE confirmation of plasmid pSB1C3-gfp-luxI construction

pSB1C3-sfgfp-luxI

This plasmid consists of two parts of target genes: sfgfp and luxI, and both of them are regulated by quorum sensing promoter. sfGFP gene is included into this plasmid as a parallel experiment group of pSB1C3-gfp-luxI. High copy number. (Fig.7-2)

Fig.7-2 AGE confirmation of plasmid pSB1C3-sfgfp-luxI construction

p3H-GFP-luxI

This plasmid consists of two parts of target genes: gfp and luxI, and both of them are regulated by quorum sensing promoter. Middle copy number. (Fig.7-3)

Fig.7-3 AGE of p3H-gfp construction

p3H-sfGFP-luxI

This plasmid consists of two parts of target genes: sfgfp and luxI, and both of them are regulated by quorum sensing promoter. sfGFP gene is included into this plasmid as a parallel experiment group of pSB1C3-gfp-luxI. Middle copy number. (Fig.7-4)

Fig.7-4 AGE of p3H-sfgfp construction

pSB3T5-aiiA

This plasmid has one target gene aiiA under the regulation of quorum sensing promoter. Middle copy number. (Fig. 7-5)

Fig.7-5 AGE confirmation of plasmid pSB3T5-aiiA construction

pSB4K5-ndh

This plasmid has one target gene aiiA under the regulation of quorum sensing promoter. Low copy number. (Fig. 7-6)

Fig.7-6 AGE confirmation of plasmid pSB4K5-ndh construction

SDS-PAGE Confirmation for Plasmids

Please refer to Improvement (For a Better Glee) in Strains comparisons.

Summary & Future work

Future Work (Keep Practicing)

Oh, no, time is up for iGEM Competition, our glee has no more time to play, but pack their luggage and head for MIT. But let's see what they have made so far and what they still have to do if they really want to be a success.

Construction Have done:

Table 6-1 Plasmids we have built
No.	Plasmid	Replication Origin	Copy Number	Resistance	Size(bp)
No.	Plasmid	Replication Origin	Copy Number	Resistance	Insert	Backbone
A1	pSB1C3-gfp-luxI	pSB1C3	high (100~300)	Chloromycetin (Cm)	4052	2070)
A2	pSB1C3-sfgfp-luxI	pSB1C3	high (100~300)	Chloromycetin (Cm)	4046	2070)
A3	p3H-GFP-luxI	p3H	Middle (18~22)	Ampicillin (Amp)	4052	/
A4	p3H-sfGFP-luxI	p3H	Middle (18~22)	Ampicillin (Amp)	4046	/
B	pSB3T5-aiiA	p15A	Middle (10~12)	Tetracycline (Tet)	2135	2837
C	pSB4K5-ndh	pSC101	Low (5)	Kanamycin (Kan)	2658	3004

Need to do:

Microfluidic

Have done:Finished building two different arrays, find basic testing parameters.

To do:Confirm more suitable microfluidic parameters for oscillation.

Strain

Have done:Confirmed DH5a's incompetent in express oscillation;

To do:Make a comparison between BL21 (WT) and BL21 (DE3) in SDS-PAGE and so on.

Copy number

Have done:Have done: Compared the arrangements of high-middle-low copy number and middle-middle-low copy number's effect on oscillation.

To do:Try other arrangements..

Fast Degrading Tag

Have done:Found LVA-tag is not fast enough for our oscillation circuit.

To do:Try LAA or other fast degrading tag for comparison.