Biobrick Transformation (6.24-6.30)

2013/9/26 20:16:39	We rehydrated the registry DNA biobricks including 13-P1-12D, 13-P1-12F, 12-P2-21F, 13-P3-11H, and 12-P4-4A. Then we transformed them into BL21. After restriction analysis of the plasmids, we prepared glyoerol stocks for BL21 with 12D, 12F, and 21F.
2013/9/26 20:17:26	M:Marker DL500 Lane1:12D plasmid Lane2:12D plasmid, digestion of EcoRI,SpeI Lane3:12D plasmid, digestion of XbaI,PstI Lane4:12F plasmid Lane5: 12F plasmid, digestion of EcoRI,SpeI Lane6: 12F plasmid, digestion of XbaI,PstI Lane7:21F plasmid Lane8:21F plasmid,digestion of EcoRI,SpeI Lane9:21F palsmid, digestion of XbaI,PstI

Plasmids Construction (7.1-7.28, 8.15-9.16)

2013/9/26 20:36:58	We purified the plasmids of 11H and 12D, after restriction analysis confirm, we inserted 11H into 12D. Next, we transformed 11H 12D into bacteria and got the plasmids to confirm. By inserting 21F into 11H 12D, we got one of our three plasmids. Finally, by using restriction analysis, we confirmed that the plasmid is right.
2013/9/26 20:37:10	M:Marker 1000bp Lane1,6,9:11H 12D 21F plasmid Lane2,4,7:11H 12D 21F plasmid,digestion of EcoRI Lane3,5,8:11H 12D 21F plasmid,digestion of EcoRI and SpeI

2013/9/26 20:38:17	We changed 12F's backbone from pSB1C3 to pSB3T5 and pSB1T3.
2013/9/26 20:38:31	M:Marker 500bp Lane1-10:colony PCR products of 12F 3T5
2013/9/26 20:39:10	M:Marker 1000bp Lane1:pSB3T5 aiiA plasmid Lane2:pSB3T5 aiiA EcoRI and SpeI digest Lane3:pSB3T5 aiiA XbaI and PstI digest

2013/9/26 20:40:27	We got ndh via PCR, and ligated it with LVA tag, but the ligation of plasmids and new backbones was failed. To solve this problem, we used new ndh whose original backbone is pSB1A2, and inserted 21F into it. The construction of ndh with pSB1K3, pSB1C3, and pSB4K5 finally finished on July 25th
2013/9/26 20:40:37	M:Marker 500bp Lane1-3:Fusion PCR products of ndh and lva-T
2013/9/26 20:42:21	M:Marker 500bp Lane1:pSB4K5 ndh plasmid Lane2:pSB4K5 ndh EcoRI digest Lane3:pSB4K5 ndh EcoRI and SpeI digest

2013/9/26 20:43:20	By protein confirm, we found that the protein our bacteria with GFP conveyed was incorrect. It was very difficult to make sure what happened to former steps. So we had to reconstruct plasmid GFP.
2013/9/26 20:43:33	M: Marker 1kb Lane1: 21F 11H 12D, digestion of EcoRI and SpeI Lane2: pSB1A3, digestion of EcoRI and XbaI
2013/9/26 20:44:08	M: Marker 1kbp Lane1, 4: pSB1A3 Lane2, 5: 21F 11H 12D Lane3, 6: aiiA

2013/9/26 20:45:14	We got sfGFP plasmid from Peking University, and to construct a complete plasmid we need, we would add a LVA tag behind sfGFP's sequence, and then construct 21F sfGFP-LVA 12D with pSB1C3 backbone.
2013/9/26 20:45:33	M1: Marker 500bp M2:Marker DL500bp Lane 1,2: 12D, digestion of EcoRI and XbaI Lane 3,4: sfGFP LVA, fusion PCR Lane 5: sfGFP, PCR Lane 6: LVA, PCR
2013/9/26 20:46:05	M: Marker 1kbp Lane1,4: 21F sfGFP LVA 12D, plasmid Lane2,5: 21F sfGFP LVA 12D, digestion of EcoRI Lane3,6: 21F sfGFP LVA 12D, digestion of EcoRI and PstI

2013/9/26 20:47:15	To make out circuit oscillate, we decided to change the backbone of GFP plasmid one more time. This time, according to reference, we needed to ligated GFP part with colE1.
2013/9/26 20:47:24	M:Marker 1kb Lane1:3H plasmid Lane2:3H plasmid,digestion of EcoRI Lane3:3H plasmid,digestion of EcoRI and SpeI.
2013/9/26 20:48:03	M:Marker 1kb Lane1,4:3H sfGFP plasmid Lane2,5:3H sfGFP plasmid,digestion of XbaI Lane3,6:3H sfGFP of XbaI and PstI Lane7,10:3HGFP plasmid Lane8,11:3H GFP plasmid,digestion of XbaI Lane9,12:3H GFP plasmid,digestion of XbaI and PstI

2013/9/26 20:48:50	To solve the problem that plasmids could not work correctly because of the bad compatibility of backbones, we decided to insert ndh part into aiiA plasmid.
2013/9/26 20:49:00	M:Marker 1kb Lane1,6:aiiAndh/3T5 plasmid,digestion of XbaI and PstI Lane2,5:aiiAndh/3T5 plasmid,digestion of XbaI Lane3,4:aiiAndh/3T5 plasmid Lane7,12:aiiA ndh plasmid,digestion of XbaI and PstI Lane8,11:aiiA ndh plasmid,digestion of XbaI Lane9,10:aiiA ndh plasmid

Transformation (7.29-9.20)

2013/9/26 20:50:04	Because we had constructed three plasmids we need, our next goal was to transform the plasmids into BL21(DE3). We first constructed a double-plasmid BL21: GFP aiiA, and then we transformed the third plasmid into BL21 with two plasmid.
2013/9/26 20:50:14	M:Marker 1kb Lane1:GFP plasmid,digestion of EcoRI and SpeI Lane2:GFP plasmid,digestion of EcoRI Lane3:GFP plasmid Lane4:aiiA plasmid Lane5:aiiA plasmid,digestion of EcoRI Lane6:aiiA plasmid,digestion of EcoRI and SpeI Lane7:ndh plasmid,digestion of EcoRI and SpeI Lane8:ndh plasmid,digestion of EcoRI Lane9:ndh plasmid Lane10,15,16:aiiA GFP plasmid Lane11,14,17:aiiA GFP plasmid,digestion of EcoRI Lane12,13,18:aiiA GFP plasmid ,digestion of EcoRI and SpeI
2013/9/26 20:51:15	M：Marker 1kb Lane1: plasmids (GFP aiiA) Lane2: three plasmids (GFP, aiiA, ndh)

2013/9/26 20:51:47	Although we have transformed three plasmids into BL21, the result was undesirable. We found that DH5α may be more suitable for our vector, so we decided to transform the plasmids into DH5α one more time. Later we constructed 1A3 GFP, colE1 GFP, sfGFP, colE1 sfGFP, and aiiA/ndh pSB3T5, so we transformed all these plasmids into DH5α for our new work of construction.
2013/9/26 20:52:17	M: Marker 1kb Lane1,6,7: GFP aiiA ndh, digestion of EcoRI and PstI Lane2,5,8: GFP aiiA ndh, digestion of EcoRI Lane3,4,9: GFP aiiA ndh plasmids

2013/9/26 20:53:01

Considered the failure of conveying in BL21, we used other bacteria for our vector. In addition, this time we transformed aiiA/ndh pSB3T5, sfGFP, colE1 GFP, colE1 sfGFP and GFP pSB1A3 besides other plasmid we had transformed into BL21.

2013/9/26 20:53:09

M:Marker 1kb Lane1,4 :GFP(1A3) aiiA ndh plasmid Lane2,5 : GFP(1A3) aiiA ndh plasmid,digestion of XbaI Lane3,6 : GFP(1A3) aiiA ndh plasmid,digestion of XbaI and PstI Lane7: GFP(3H) aiiA ndh plasmid Lane8: GFP(3H) aiiA ndh plasmid digestion of XbaI Lane9: GFP(3H) aiiA ndh plasmid digestion of XbaI and PstI Lane10: sfGFP(1C3) aiiA ndh plasmid Lane11: sfGFP(1C3) aiiA ndh plasmid digestion of XbaI Lane12: sfGFP(1C3) aiiA ndh plasmid digestion of XbaI and PstI Lane13: sfGFP(3H) aiiA ndh plasmid Lane14: sfGFP(3H) aiiA ndh plasmid digestion of XbaI Lane15: sfGFP(3H) aiiA ndh plasmid digestion of XbaI and PstI

2013/9/26 20:56:19

Considered the failure of conveying in BL21, we used other bacteria for our vector. In addition, this time we transformed sfGFP, colE1 GFP, and colE1 sfGFP, GFP pSB1A3 besides other plasmid we had transformed into BL21.

SDS-PAGE

2013/9/26 21:49:44

W: Whole cell P: Precipitation S: Supernatant

2013/9/26 21:51:07

2013/9/26 21:55:45

2013/9/26 22:03:34

choose the photo you want to Description: We compared protein bands between BL21, the blank control and three groups transformed into different single plasmids, aiiA, ndh and gfp. These two gel pictures were got in two different days. Data analysis: The target proteins of pSB3T5-aiiA/BL21 and pSB4K5-ndh/BL21 should not be expressed because the promoter should be induced by AHL. The target proteins of pSB1C3-gfp-luxI/BL21 has been expressed but GFP was misfolded we suppose. However, the band marked in red was not the target band. Maybe it's the complex of AHL-Lux

2013/9/26 22:07:07

choose the photo you want to Description: We performed a SDS-PAGE comparing expression of double plasmids, gfp and aiiA, and two corresponding single plasmids. What's more, we want to find more details about band which is possible the misfolded gfp. Data analysis: The band marked in red disappeared is pSB1C3-gfp-luxI/BL21.The LuxR, GRP and AiiA are the same of size (marked in yellow). However the band of AiiA was not clear. Many target proteins were misfolded. Therefore the culture condition should be optimized.

2013/9/26 22:08:04

Characterization of expression of E.coli transformed into triple plasmids and testing if our target proteins expressed by our constructed gene circuits were concentrated in precipitation or supernatant (8.12)

2013/9/26 22:07:46

Description : We had transformed triple plasmids into one E.coli and the DNA gel picture indicated it was ligated successfully. So know we were very eager to know if our circuits could work well in E.coli. Furthermore, we wanted to know that if our target proteins expressed by our constructed gene circuits, in other words, pSB1C3-gfp-luxI and pSB13T5-aiiA were aggregated in precipitation or supernatant. So we run SDS-PAGE using BL21(blank control), three cells transformed into different single plasmids(aiiA, ndh and gfp), one cell transformed into double plasmids(aiiA and gfp) and our E.coli transformed into triple plasmids in three forms respectively, whole cell, precipitation and supernatant. Data analysis: From the SDS-PAGE gel pictures, we could easily find that the clear AiiA band (marked in red) but NDH was not clear in PAGE because the gene ndh does exist in the genome DNA of BL21 native strain.

2013/9/26 22:08:47

choose the photo you want to Description: We've found that maybe there's some mistakes in our strains which cause our couldn't get a expected microfluidic experiment result. And we analyzed that DH5α maybe a better strain than BL21 so we change the host. We firstly confirm the expression of three single plasmids transformed into DH5α. Data analysis: The band marked in red was not the target band. Maybe it is the complex of AHL-LuxI. The target proteins of pSB1C3-gfp-luxI has been expressed while the target proteins of pSB3T5-aiiA and pSB4K5-ndh did not be expressed because the promoter should be induced by AHL.

2013/9/26 22:09:28

choose the photo you want to Description: We were suggested that DH5α has a better viability and a higher protein expression. So we maybe there's some mistakes in our strains which cause our couldn't get a expected microfluidic experiment result. So we compared two identical groups cultured at different degrees, 30℃ and 37℃. Data analysis: E.coli maybe cultured at 30℃ has a stronger expression of protein from picture 1 but we were confused by picture 2 cause it indicated that expression at 30℃ is not larger than expression at 37℃.

2013/9/26 22:09:40

Future plan: 1.It's much better to culture at 30℃ rather than 37℃. 2.Does it better for us to use superfolded GFP? 3.Change another ultrasonic instrument or high pressure homogenizer.

2013/9/26 22:10:54

2013/9/26 22:11:28

Description: We changed high pressure homogenizer to get cell fragments, in order to gain a more definite gel analysis. The high pressure homogenizer is under 37.5 kpSI which supposed much too high to make protein. Even the super protein is far less compared with gel picture made after ultrasonic fragmentation, the unclear band is still there. Data analysis: The band marked in red was not the target band. The band marked in green was not the target band.

2013/9/26 22:12:23

2013/9/26 22:12:32

Description: We changed high pressure homogenizer to get cell fragments, in order to gain a more definite gel analysis. The high pressure homogenizer is under 37.5 kpSI which supposed much too high to make protein. Even the super protein is far less compared with gel picture made after ultrasonic fragmentation, the unclear band is still there. Data analysis: In picture 1, the band marked in red was not the target band. In picture 2, the band marked in red disappeared is pSB1C3-gfp-luxI/DH5α and pSB3T5-aiiA/DH5α.

2013/9/26 22:13:16

Description: We were confused for our dislocation of GFP on SDS-PAGE gel pictures for a long time. So we decided to use three different marker to confirm our marker. Data analysis: There is a paradox between A A and A: E.coli cultured has a stronger expression of protein when not added AHL？

Microfluidic Experiment Record

2013/9/25 20:38:47

8th August Plasmid:Gfp(pSB1C3) aiia(pSB3T5) Bacteria:BL21(DE3) Condition: Supply medium culture is not flowing.. Cells trap in the micro pools. Without antibiotic putting in. Total Obserbation Time:200mins Capturing time lapse:5mins Analysis: At the beginning, the population of cells is small and the fluorescence degree is as the same. Minutes later, more cells grew as well as stronger fluorescence was detected. From then on, we observed weak oscillation of the fluorescence intensity.

2013/9/25 20:42:38

9th August Plasmid:Gfp(pSB1C3) aiia(pSB3T5) Bacteria:BL21(DE3) Condition: Supply medium culture is not flowing.. Cells trap in the micro pools. With antibiotic putting in. Total Obserbation Time:140mins Capturing time lapse:5mins Analysis: At the beginning, the population of cells is small and the fluorescence degree is as the same. Minutes later, more cells grew but the population is still not large enough to observe convictive fluorescence intensity changing.

2013/9/25 20:45:15

25th August Plasmid:Gfp(pSB1C3) aiia(pSB3T5) Bacteria:DH5a Condition: Supply medium culture is not flowing.. Cells trap in the micro pools. With antibiotic putting in. Total Obserbation Time:165mins Capturing time lapse:5mins Analysis: At the beginning, the population of cells is small and the fluorescence degree is as the same. Minutes later, more cells grew and we detect stronger fluorescence degree. From then on, the fluorescence degree kept in almost the same intensity.

2013/9/25 20:46:04

31th Auguest Plasmid:Gfp(pSB1C3) aiia(pSB3T5) Bacteria:DH5a Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Total Obserbation Time:80mins Capturing time lapse:5mins Analysis: At the beginning, the population of cell was large enough to get valid fluorescence degree. And from then on , we observed no fluorescence intensity changing.

2013/9/25 20:47:42

12th September Plasmid:Gfp(pSB1C3) aiia(pSB3T5) Bacteria:DH5a Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Total Obserbation Time:180mins Capturing time lapse:5mins Analysis: At the beginning, the population of cell was large enough to get valid fluorescence degree. From then on ,there was a weak decrease of the fluorescence degee.

2013/9/25 20:50:35

15th September Plasmid:Gfp(pSB1C3) aiia(pSB3T5) Bacteria: BL21(WT) Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Total Obserbation Time:515mins Capturing time lapse:5mins Analysis: From all the mircrofluid images, there is a tendency that the fluorescence degree change from weak to strong and then change from strong to weak.

2013/9/25 20:52:02

18th September Plasmid:sfGfp(coE1) aiia(pSB3T5) Bacteria: BL21(WT) Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Total Obserbation Time:590mins Capturing time lapse:5mins Analysis: There was a strong tendency that the fluorescence intensity came down, and it's the first time that we detected the most obvious changing.

2013/9/25 21:11:06

20th September Plasmid:sfGfp(coE1) aiia(pSB3T5) Bacteria: BL21(WT) Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Total Obserbation Time:470mins Capturing time lapse:5mins Analysis: From all the microfluid images, there is a very weak decrease of the fluorescence degree.

2013/9/25 21:13:01

22th September Plasmid:sfGfp(coE1) aiia(pSB3T5) Bacteria: BL21(WT) Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Total Obserbation Time:? Capturing time lapse:5mins Analysis: ?

2013/9/25 21:15:47	Microscope
2013/9/25 21:16:23	Microfluidic chip
2013/9/25 21:16:51	Ports