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.
2013/9/28 01:03:53	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.
2013/9/28 01:05:10	M:Marker 500bp Lane1-10:colony PCR products of 12F+3T5
2013/9/28 01:05:15	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
2013/9/28 01:06:41	M1:Marker 500bp Lane1-3:Fusion PCR products of ndh and lva-T
2013/9/28 01:06:51	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.
2013/9/28 01:10:10	M: Marker 1kb Lane1: 21F+11H+12D, digestion of EcoRI and SpeI Lane2: pSB1A3, digestion of EcoRI and XbaI
2013/9/28 01:10:38	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.
2013/9/28 01:11:54	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
2013/9/28 01:12:42	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 our 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.
2013/9/28 01:16:48	M:Marker 1kb Lane1:3H plasmid Lane2:3H plasmid,digestion of EcoRI Lane3:3H plasmid,digestion of EcoRI and SpeI.
2013/9/28 01:17:27	M:Marker 1kb Lane1,4:3H+sfGFP plasmid Lane2,5:3H+sfGFP plasmid,digestion of XbaI Lane3,6:3H+sfGFP of XbaI and PstI Lane7,10:3H+GFP 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.
2013/9/28 01:19:02	M:Marker 1kb Lane1,6:aiiA/ndh+3T5 plasmid,digestion of XbaI and PstI Lane2,5:aiiA/ndh+3T5 plasmid,digestion of XbaI Lane3,4:aiiA/ndh+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 plasmids.
2013/9/28 01:21:56	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
2013/9/28 01:22:37	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.
2013/9/28 01:23:53	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.

2013/9/28 01:24:59

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

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 have 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-LuxI.

2013/9/26 22:07:07

We performed a SDS-PAGE comparing expression of double plasmids, gfp and aiiA, and two corresponding single plasmids. What's more, we wanted to find more details about band which possibly is the misfolded gfp. Data analysis: The band marked in red disappeared is pSB1C3-gfp-luxI/BL21.The LuxR, GFP 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 now 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 did exist in the genome DNA of BL21 native strain.

2013/9/26 22:08:47

We've found that maybe there's some mistakes in our strains which cause us couldn't get a expected microfluidic experiment result. And we analyzed that DH5α maybe a better strain than BL21 so we changed 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 have 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

We were suggested that DH5α has a better viability and a higher protein expression. So there's some mistakes in our strains which cause us 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℃ had 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？

2013/9/28 07:03:54

Description: We characterize our biobricks using additional AHL. We bought commercial AHL powder and prepared it into solution. The AHL added into these cells was 1000 nmol/L and BL21 was wild type. Data analysis: The band marked in yellow disappeared. There is a difference between N and N+A： The band marked in green/red shows that E.coli cultured has a stronger expression of protein when added AHL?

2013/9/28 08:44:22	We continued to characterize our biobricks using additional AHL. These plasmids included sfgfp, sfgfp-aiiA, sfgfp-aiiA-ndh. SDS-PAGE gel pictures are shown as follows. Illustration: A:BL21(WT) B:BL21(WT) 1C3-sfgfp C:BL21(WT) 1C3-sfgfp-aiiA D:BL21(WT) 1C3-sfgfp-aiiA-ndh E:3H-1C3-sfgfp-aiiA-ndh
2013/9/28 08:47:11	We continued to characterize our biobricks using additional AHL. These plasmids included sfgfp, sfgfp aiiA, sfgfp aiiA ndh. SDS-PAGE gel pictures are shown as follows. Illustration: A:BL21(WT) B:BL21(WT) 1C3-sfgfp C:BL21(WT) 1C3-sfgfp-aiiA D:BL21(WT) 1C3-sfgfp-aiiA-ndh E:3H-1C3-sfgfp-aiiA-ndh
2013/9/28 08:44:24	We continued to characterize our biobricks using additional AHL. These plasmids included sfgfp, sfgfp aiiA, sfgfp aiiA ndh. SDS-PAGE gel pictures are shown as follows. Illustration: A:BL21(WT) B:BL21(WT) 1C3-sfgfp C:BL21(WT) 1C3-sfgfp-aiiA D:BL21(WT) 1C3-sfgfp-aiiA-ndh E:3H-1C3-sfgfp-aiiA-ndh
2013/9/28 08:44:24	We continued to characterize our biobricks using additional AHL. These plasmids included sfgfp, sfgfp aiiA, sfgfp aiiA ndh. SDS-PAGE gel pictures are shown as follows. Illustration: A:BL21(WT) B:BL21(WT) 1C3-sfgfp C:BL21(WT) 1C3-sfgfp-aiiA D:BL21(WT) 1C3-sfgfp-aiiA-ndh E:3H-1C3-sfgfp-aiiA-ndh
2013/9/28 08:44:28	Description: We continued to characterize our biobricks using additional AHL. These plasmids included sfgfp, sfgfp aiiA, sfgfp aiiA ndh. SDS-PAGE gel pictures are shown as follows. Illustration: A:BL21(WT) B:BL21(WT) 1C3-sfgfp C:BL21(WT) 1C3-sfgfp-aiiA D:BL21(WT) 1C3-sfgfp-aiiA-ndh E:3H-1C3-sfgfp-aiiA-ndh

Microfluidic Experiment Record

2013/9/25 20:38:47

8th August Plasmid:gfp(pSB1C3)+aiiA(pSB3T5) Bacteria:BL21(DE3) Condition: No flowing medium culture supply. Cells trap in the micro pools. Without antibiotic putting in. Capturing time lapse:5mins Total Obserbation Time:200mins Analysis: At the beginning, the population of cells was small and the fluorescence degree was 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

choose the photo yo9th August Plasmid:gfp(pSB1C3)+aiiA(pSB3T5) Bacteria:BL21(DE3) Condition: No flowing medium culture supply. Cells trap in the micro pools. With antibiotic putting in. Capturing time lapse:5mins Total Obserbation Time:140mins Analysis: At the beginning, the population of cells was small and the fluorescence degree was as the same. Minutes later, more cells grew but the population was 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: No flowing medium culture supply. Cells trap in the micro pools. With antibiotic putting in. Capturing time lapse:5mins Total Obserbation Time:165mins Analysis: At the beginning, the population of cells was small and the fluorescence degree was as the same. Minutes later, more cells grew and we detected 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. Capturing time lapse:5mins Total Obserbation Time:80mins 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. Capturing time lapse:5mins Total Obserbation Time:180mins 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 degree.

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. Capturing time lapse:5mins Total Obserbation Time:515mins Analysis: From all the mircrofluid images, there was a tendency that the fluorescence degree changed from weak to strong and then changed from strong to weak.

2013/9/25 20:52:02

18th September Plasmid:sfgfp(colE1)+aiiA(pSB3T5) Bacteria: BL21(WT) Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Capturing time lapse:5mins Total Obserbation Time:590mins 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(colE1)+aiiA(pSB3T5) Bacteria: BL21(WT) Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in. Capturing time lapse:5mins Total Obserbation Time:470mins Analysis: From all the microfluid images, there was a very weak decrease of the fluorescence degree.

2013/9/25 21:13:01	4X
2013/9/28 07:14:25	10X
2013/9/28 07:14:27	20X(light field)
2013/9/28 07:14:28	20X 22th September Plasmid:sfgfp(colE1)+aiiA(pSB3T5) Bacteria: BL21(WT) Condition: Flowing medium culture supply. Cells could flow into different traps. With antibiotic putting in.Changing different lens magnification Total Obserbation Time:600mins Observe time interval:20mins Analysis: There was a tendency that the fluorescence degree changed from weak to strong and then changed from strong to weak.

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

RFU (9.24-9.27)

2013/9/27 21:59:17	As we have made BL21(WT) with several plasmids, it was necessary to determine the OD and fluorescence value of the bacteria so that we could know if BL21(WT) with the plasmids was able to oscillate. To achieve the goal, we used enzyme-labeled instrument. By sampling and measuring on time, we could see that if our circuit would work smoothly. After sampling per hour, we centrifuged samples at 6,000g for 10min. And then we discarded flow-through liquid. Next, we added PBS buffer into bacteria. Using enzyme-labeled instrument, we got a serious of data of OD and fluorescence value. We did it three times.
2013/9/28 03:11:44	The first time we measured the fluorescence value.
2013/9/28 03:30:57	The second time we measured the fluorescence value.
2013/9/28 03:31:38	The second time we measured the OD.
2013/9/28 03:34:06	The third time we measured the fluorescence value.