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Plasmid Plug&Play

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Associative Memory
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Contents 1 Lab Diary 1.1 Plasmid Plug&Play 1.2 Associative Memory Network 1.2.1 1- Greg's Lab Experiments 1.2.2 General experiments 1.2.3 Assembly experiments 1.2.3.1 MR2 1.2.3.2 MR3 1.2.3.3 MR6 1.2.3.4 MR7 1.2.3.5 MR9 1.2.4 2- Hotta's and Cleslei's Lab experiments 1.2.5 Experiments of Biobricks 1.2.6 Assembly experiments 1.2.7 From 12/06/2012 to 12/08/2012 – Assemblies attempts 1.2.8 First assemblies confirmed 1.2.8.1 QC1 1.2.8.2 QC4 1.2.8.3 QC5 1.2.8.4 QC6 1.2.8.5 QR4 1.2.8.6 QR5 1.2.8.7 QR6 1.2.8.8 QR1

Lab Diary

Plasmid Plug&Play

The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.

Plug&Play Experiments

Associative Memory Network

1- Greg's Lab Experiments

General experiments

17/07/12 – Amanda and Cleandho: - Tetracicline test using TOP10 without plasmid: successful, antibiotic is working

18/07/12 – Aline and Cleandho: - Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter

02/08/12 – Cleandho and Lucas: - Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use. - Make competent cells for storage and use in -80°C

03/08/12 – Cleandho and Lucas: - Competent cells test for viability and competency

22/08 – Cleandho and Lucas: - Make competent cell for use and storage at -80°C - Viability and competency tests of competent TOP 10

23/08/12 – Débora: - Digestion of pSB1K3 with PstI and EcoRI

03/09/12 – Cleandho: - Preparation of LB culture media

05/09/12 – Cleandho and Débora: - Digestion of pSB1K3, pSB1C3

Assembly experiments

MR2

09/07/12 - Amanda, Cleandho, Débora and Lucas: - Bacterial transformation with MR2 part. MR2 grew colonies

10/07/2012 - Amanda, Cleandho, Débora and Lucas: - Bacterial clones from MR2 were inoculated in LB with tetracycline

11/07/2012 – Amanda, Cleandho, Débora and Lucas: - Plasmid extraction of MR2 and quantification by nanodrop Expected size: MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb - Digestion with enzymes and incubate 37 ° C for 1 hour MR2 – (XbaI and PstI)

11/07/2012 – Aline and Cleandho: - Electrophoresis to confirmation: no DNA yield

17/07/12 – Amanda and Cleandho: - Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell 18/07/12 – Aline and Cleandho: - Ligation of the parts MR2 using pSB1C3 backbone

19.07.12 – Aline, Cleandho and Débora: - Transformation of MR2

23/07/12 – Cleandho and Débora: - Quantification of pDNA: MR2-2: 119,9 ng/uL - Digestion, using approximately 300ng of DNA MR2: XbaI + PstI

24/07/12 – Cleandho: - Electrophoresis to confirmation of MR2-1 andMR2-2 clones: MR2-2 is positive and MR2-1 was negative.

 

MR3

09/07/12 - Amanda, Cleandho, Débora and Lucas: - Bacterial transformation with MR3 part. MR3 – grew colonies

10/07/2012 - Amanda, Cleandho, Débora and Lucas: - Bacterial clones from MR3 were inoculated in LB with tetracycline

11/07/2012 – Amanda, Cleandho, Débora and Lucas: - Plasmid extraction of MR3 Expected size: MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb - Digestion with enzymes and incubate 37 ° C for 1 hour MR3 – ( EcoRI and SpeI)

11/07/2012 – Aline and Cleandho: - Electrophoresis to confirmation: no DNA yield

18/07/12 – Aline and Cleandho: - Ligation of the part MR3 using pSB1C3 backbone

19.07.12 – Aline, Cleandho and Débora: - Transformation of MR3.

23/07/12 – Cleandho and Débora: - Quantification of pDNA: MR3-1: 110,8 ng/uL - Digestion, using approximately 300ng of DNA MR3: EcoRI + SpeI

24/07/12 – Cleandho: - Electrophoresis to confirmation of MR3-1 clone: no DNA yield

07/08/12 - Cleandho and Lucas: - Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3

09/08/12 – Amanda - Transformation of MR3 assembly

14/08/12 – Lucas and Amanda - Plasmid extraction from two MR3 clones - Digestion of MR3 clones with (EcoRI + SpeI) - Electrophoresis to confirm MR3 clones: result failed

22/08 – Cleandho and Lucas: - Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) - Transformation of MR3 assembly

04/09/12 – Cleandho: - Plasmid extraction of six MR3 clones - Digestion of MR3 clones with EcoRI and SpeI - Quantification of DNA of MR3 clones: A 178 ng/uL B 129 ng/uL C 119 ng/uL D 98 ng/uL E 159 ng/uL F 110 ng/uL - Digestion of MR3 using HindIII - Electrophoresis of MR3 digested with HindIII. Result: all clones have failed

05/09/12 – Cleandho and Débora: - Digestion of pSB1K3, pSB1C3, 2M, 4G - Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3

06/09/12 – Cleandho: - Transformation of MR3 assembly in three molar proportions.

11/09/12 – Cleandho and Lucas: - PCR screening of ten colonies clones of assembly MR3

12/09/12 – Cleandho and Lucas: - Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon - Inoculum of clone 4 of MR3 in LBK

13/09/12 Cleandho and Lucas: - Plasmid extraction of clone 4 from MR3 assembly - Digestion of pDNA of clones 4 using EcoRI and SpeI - Electrophoresis for confirmation of clone 4. Result: failed

 

MR6

09/07/12 - Amanda, Cleandho, Débora and Lucas: - Bacterial transformation MR6 part. MR6 – not grown colonies

17/07/12 – Amanda and Cleandho: - Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell

18/07/12 – Aline and Cleandho: - Ligation of the part MR6 using pSB1C3 backbone

19.07.12 – Aline, Cleandho and Débora: - Transformation of MR6

23/07/12 – Cleandho and Débora: - Quantification of pDNA: MR6-1: 45,6ng/uL MR6-5: 46,7ng/uL - Digestion, using approximately 300ng of DNA MR6: EcroRI + SpeI

24/07/12 – Cleandho: - Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed

02/08/12 – Cleandho and Lucas: - Ligation/Assembly 3A of MR6 part

03/08/12 – Cleandho and Lucas: - Transformation of MR6

08/08/12 – Débora: - Plasmid extraction from MR6 clones - Digestion of pDNA with EcoRI + PstI from MR6 clones

09/08/12 – Amanda - Electrophoresis to confirm MR6 clones. Result: failed

03/09/12 – Cleandho: - Preparation of LB culture media - Inoculum of six MR6 clones

05/09/12 – Cleandho and Débora: - Digestion of pSB1K3, pSB1C3, 11J and 16P - Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3

06/09/12 – Cleandho: - Transformation of MR6 assembly in three molar proportions.

12/09/12 – Cleandho and Lucas: - Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct  

MR7

14/08/12 – Lucas: - Ligation of assembly MR7 using pSB1K3

24/08/12 – Débora: - Transformation of MR7 assembly

28/08/12 – Débora and Lucas: - Plasmid extraction of MR7 clones - Digestion of MR7 clones with XbaI and PstI

29/08/12 – Débora and Lucas: - Electrophoresis of two clones of MR7 assembly. Result: failed  

MR9

14/08/12 – Amanda - Transformation of MR9 assembly

27/08/12 – Lucas: - Plasmid extraction for MR9 clones - Digestion of MR9 clones with EcoRI and SpeI - Electrophoresis of MR9 clones. Result: failed

29/08/12 – Débora and Lucas: - Transformation of MR9 assembly

2- Hotta's and Cleslei's Lab experiments

Experiments of Biobricks

At this first moment, we worked on the confirmation of every biobrick that should be used in the project. We transformed and analyzed all the biobricks, using minipreping, nanodroping, digestion and electrophoresis gel. This time was reserved to train everybody in lab techniques, testing protocols and adjusting of the project.

The follow biobricks were confirmed along April and May. On Hotta's Lab participated Lilian, Otto, Daniel, Pedro and Luiza. On Cleslei's Lab, only Fernando

We had issues in the confirmation of small parts (<100 kb), due to the resolution of gel. After this period, we shared the assemblies that should be done in order to start the tests of the system.

Assembly experiments

From 12/06/2012 to 12/08/2012 – Assemblies attempts

At this period, the first attempts in order to construct the lineages that should interact in the system were made. We tried the assemblies QC1, QC6, QR4, MR1 and MR2 (see the full diagram HERE). Nothing worked properly, we had lots of fake positives or assemblies’ transformations that didn’t had any colonies. In order to fix it, we tested every single step of 3A protocols, modifying the proportions of buffer, ligase, vector and parts, assembly time, temperatures and etc. These procedures consumed a big part of our time and resources and the difficulties retarded our advancement, especially on vacations, time when we intent to accelerate the assemblies.

At the end of all tests, we discovered that the enzyme and buffers were, somehow, inadequate. Were made attempts with different brands of buffer and enzymes and a new T4 ligase showed itself as good choice.

First assemblies confirmed

After this messy period trying to fix the assembly's problems, the good results came. We made with fully success QC1, QC4, QC5, QR4, QR5 and QR1 assemblies through 3A protocol. The results and Agarose Gel photos follows.

QC1

12/06/2012 - Lilian, Ligation

13/06/2012 - Daniel, Transformation

14/06/2012 - Daniel, Inoculate

15/06/2012 - Daniel, Miniprep

16/06/2012 - Otto, Digestion

17/06/2012 - Daniel, Electrophoresis Gel

QC4

06/08/2012 - Pedro, Ligation

07/08/2012 - Pedro, Transformation

10/08/2012 - Daniel, Inoculate

11/06/2012 - Daniel, Miniprep

13/06/2012 - Pedro, Digestion

17/06/2012 - Pedro, Electrophoresis Gel

QC5

14/08/2012 - Pedro, Ligation

15/08/2012 - Daniel, Transformation

17/08/2012 - Pedro & Daniel, Miniprep, Digestion and PCR

18/08/2012 - Daniel, Electrophoresis Gel

QC6

12/06/2012 - Lilian, Ligation

13/06/2012 - Daniel, Tranformation

14/06/2012 - Daniel, Inoculum

15/06/2012 - Otto, Miniprep

16/06/2012 - Otto, Digestion

17/06/2012 - Daniel, Electrophoresis Gel

QR4

14/05/12 – Fernando: Bacterial transformation with 12C (QR4), 12H (QR1) and 14A (QR4) parts. Bacterial clones were plated on LB plates with appropriate antibiotic and grown overnight at 37C.

15/05/12 – Fernando: Bacterial clones were inoculated in 5mL LB with appropriate antibiotic and grow overnight at 37C.

16/05/12 – Fernando: 500ul from each inoculate was plated on LB plates with appropriate antibiotics. The remaining volume was used for DNA extraction, using Quiagen Miniprep Kit.

17/05/12 – Fernando: Bacterias carrying biobricks 12C, 12H and 14A, were frozen on 20% glycerol at -80C for further use. Parts 12H and 14A were confirmed after gel electrophoresis.

03/06/12 – Fernando: Three transformations were made using 50ul TOP10 bacteria and 2uL miniprep volume from 1D (QR3), 2M (QR2) and 12M (QR1) parts and plated on LB with appropriate antibiotics.

04/06/12 – Fernando: Single colonies were inoculated in 5mL LB + antibiotics.

05/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.

06/06/12 – Fernando: The 500ul plated on the day before (1D, 2M and 12M) were frozen in 20% glycerol at -80C.

09/06/12 – Fernando: Two transformations were performed with 50ul TOP10 bacteria using 2uL miniprep from pSB1C3 and 18K (QR5).

10/06/12 – Fernando: Single colonies from pSB1C3 and 18K were inoculated in 5mL LB + antibiotics.

11/06/12 – Fernando: 4,5mL from each inoculate was used for DNA extraction, the remaining 500uL was plated on LB with appropriate antibiotics.

11/06/12 - Fernando: 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).

12/06/12 – Fernando: All colonies were red, showing that the assembly did not work.

14/06/12 – Fernando: 3A Assembly Protocol was used to assemble 12H and 12C using pSB1C3 as plasmid backbone and also 12C and 14A, with pSB1C3 as backbone (QR4). This time newer enzymes were used.

15/06/12 – Fernando: All colonies were still red, showing that the assembly did not work.

QR5

01/08/12 – Fernando: 3A Assembly Protocol was used to perform QR4 + 18K + pSB1C3 (QR5) assembly. A few changes were made to this protocol. - The enzyme NotI was used instead of DpnI. - The ligation step was performed using 4uL of digested plasmid backbone and not 2uL. - 2uL T4 DNA ligase was used instead of 1uL - No water added

02/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.

06/08/12 – Fernando: Four colonies were inoculated in 5mL LB + antibiotics and grown overnight.

07/08/12 – Fernando: Each inoculate was used for DNA extraction. 3uL plasmid from each of the four colonies were digested with EcoRI and PstI enzymes. QR5 assembly was confirmed with gel electrophoresis (three out of the 4 colonies were confirmed).

QR6

08/08/12 – Fernando: 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly.

09/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.

10/08/12 – Fernando: All colonies were red.

22/08/12 – Fernando: One more change was made in 3A Assembly Protocol. The enzyme HindIII was used instead of NotI. (This prevented the RFP coding device to ligate again to the pSB1C3 after digestion, so less red colonies were seen). 3A Assembly Protocol was used to perform QR5 + 12M + pSB1C3 (QR6) assembly.

23/08/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of the ligation step.

26/08/12 – Fernando: Five colonies were inoculated in 5 mL LB + antibiotics and grew overnight.

27/08/12 – Fernando: DNA was extracted using Quiagen Miniprep Kit. The QR6 construction was digested with BamHI and PstI, but the 2.363bp and 1.613bp bands could not be distinguished after gel electrophoresis.

01/09/12 – Fernando: QR6 construction were digested again using a different set of enzymes. 3A Assembly Protocol was used to assemble 12H and 12M using pSB1C3 as plasmid backbone (QR1).

02/09/12 – Fernando: 50uL TOP10 bacteria transformed with 2uL product of QR1 ligation step.

03/09/12 – Fernando: All colonies were red

20/09/12 – Fernando: Since 3A Assembly Protocol was not working to assemble the QR1 construct, each part (12H, 12M and pSB1C3) were digested using the 3A Assembly Protocol digestion procedure and the total volume was loaded in a electrophoresis gel. The idea was to purify the inserts and linearized plasmid backbone pSB1C3 using Quiagen Gel Extraction Kit. Since the bands could barely be seen under U.V, nothing was purified.

QR1

18/09/2012 - Pedro - Ligation.

19/09/2012 - Daniel & Luiza - Transformation.

20/09/2012 - Daniel - Inoculum.

21/09/2012 - Pedro - Digestion and PCR.

24/09/2012 - Pedro - Electrophoresis Gel.

At this moment we’re having issues on the assembly of small parts. Other methods of assembling, such as the Standard, are being tested in order to overpass it.