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| | <br> | | <br> |
| | <br> | | <br> |
| - | The fragments will be frozen and purified later. | + | The fragments will be frozen and later purified . |
| | <br> | | <br> |
| | <br> | | <br> |
| Line 158: |
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| | | | |
| - | We got a new batch of primers today. They need to be diluted and stocked, before they are ready for use. | + | We got a new delivery of primers from IDT today. They are to be diluted and stocked, before use. |
| | | | |
| | We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle. | | We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle. |
| | <br> | | <br> |
| | <br> | | <br> |
| - | <b>The PCR reactions were successful, so following biobricks are ready for Plug'n'Play: </b> | + | <b>The PCR reactions were successful, and following biobricks are ready for Plug'n'Play: </b> |
| | | | |
| | <ul> | | <ul> |
| Line 177: |
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| | <br> | | <br> |
| | <a name="Tuesday 26.07.2011"></a><h4>Tuesday</h4> | | <a name="Tuesday 26.07.2011"></a><h4>Tuesday</h4> |
| - | The new primer batch we recieved yesterday, will be used for amplification of many many many new biobricks today. | + | The primers recieved yesterday will be used for amplification of many many many new biobricks today. |
| | <br> | | <br> |
| | <br> | | <br> |
| - | <b>Out of 15 PCR reactions only 5 succeeded: </b> | + | <b>Out of 15 PCR reactions 5 succeeded: </b> |
| | | | |
| | <br> | | <br> |
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Line 200: |
| | <a name="Wednesday 27.07.2011"></a><h4>Wednesday</h4> | | <a name="Wednesday 27.07.2011"></a><h4>Wednesday</h4> |
| | | | |
| - | Today we have run 8 reaction, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining. | + | Today we have run 8 reactions, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining. |
| | <br> | | <br> |
| | <br> | | <br> |
| - | <b>Only one succeeded:</b> | + | <b>One succeeded:</b> |
| | <ul> | | <ul> |
| | <li>hgH-polyA (<b>33</b>) </li> | | <li>hgH-polyA (<b>33</b>) </li> |
| Line 215: |
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| | <a name="Week 5"></a><h3><b>Week 5: 01.08.2011 - 07.08.2011</b></h3> | | <a name="Week 5"></a><h3><b>Week 5: 01.08.2011 - 07.08.2011</b></h3> |
| | <a name="Monday 01.08.2011"></a><h4>Monday</h4> | | <a name="Monday 01.08.2011"></a><h4>Monday</h4> |
| - | New week, new energy! | + | New week, new energy, and lots of team spirit! |
| | Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic! | | Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic! |
| | <br> | | <br> |
| Line 247: |
Line 247: |
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| | <a name=" Wednesday 03.08.2011"></a><h4> Wednesday </h4> | | <a name=" Wednesday 03.08.2011"></a><h4> Wednesday </h4> |
| - | We still have 2 biobricks we haven’t been able to obtain with PCR: pyrG and pyrG-DR.
| + | There are still 2 BioBricks that we haven't been able to obtain with PCR: pyrG and pyrG-DR. |
| | <br> | | <br> |
| - | Today we tried again with gradient PCR to find the optimal melting temperature, but it did not help. | + | Today we tried again with gradient PCR to find the optimal melting temperature, but no magic happened. |
| | <br> | | <br> |
| | <br> | | <br> |
| | <a name="Thursday 04.08.2011"></a><h4>Thursday</h4> | | <a name="Thursday 04.08.2011"></a><h4>Thursday</h4> |
| - | Today we purified 22 biobricks, which have been obtained with PCR during the last week of laboratory work. The purification of DNA was done by using the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) according to the manufactures manual for purification of DNA from TAE and TBE agarose gels protocol, see protocol section for more information.<br><br>
| + | 22 BioBricks, which have been obtained with PCR during the last week of laboratory work, was purified today. The purification of DNA was done by using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The procedure was carried out according to the manufactures protocol for purification of DNA from TAE and TBE agarose gels, see protocol section for more information.<br><br> |
| - | Despite several attempts, we have not been able to obtain biobrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, meaning that the DNA template was the source of error. | + | Despite several attempts we have not been able to obtain BioBrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, and thereby it revealed that the DNA template was the source of error. Problem solved! |
| | <br> | | <br> |
| | <br> | | <br> |
| | | | |
| - | We also started cultivation of the mammalian cell line, U2OS, which is derived from a 15-year old patient with osteosarcoma (bone cancedr). They were defrosted and cultivated in in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section. | + | We also started cultivation of the mammalian cell line, U2OS, which is derived from a patient with osteosarcoma (bone cancer). They were defrosted and cultivated in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section. |
| | <br> | | <br> |
| | <br> | | <br> |
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| | <a name="Monday 08.08.2011"></a><h4>Monday</h4> | | <a name="Monday 08.08.2011"></a><h4>Monday</h4> |
| | | | |
| - | Today we made the first USER cloning, it is very exciting to see if our system works! <br> | + | Today we made the first USER cloning, we are very excited to see if the system works the way, we want it to! <br> |
| | | | |
| | All our biobricks are divided into devices, see protocol for USER MIX. <br> | | All our biobricks are divided into devices, see protocol for USER MIX. <br> |
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Lab notes
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July
Week 1: 04.07.2011 - 10.07.2011
First week! Time was spent on designing the Plug 'n' Play assembly system, figuring out what 48 biobricks to construct, and searching for appropriate DNA templates.
Week 2: 11.07.2011 - 17.07.2011
Wednesday
The first batch of primers have been ordered, so we are waiting in excitement for them to arrive, so we can start our work in the lab.
Week 3: 18.07.2011 - 24.07.2011
Tuesday
First day in the lab and 48 biobricks to go. This is exciting!
We plan to construct 48 new BioBricks. We have now established 17 of them and ordered primers for them with USER tails. We started the day with 11 PCR reactions. 6 of them were correct, and they were purified with a purification Kit (GE Healthcare). Only 42 biobricks to go!
Biobricks in the bag:
- pALC (7)
- TtrpC (13)
- Terminator 1 (14)
- Terminator 2 (15)
- Terminator 3 (16)
- argB (17)
Thursday
The failed PCR reactions from tuesday were repeated. This time we used a different annealing temperature, and....IT WORKED!
New biobricks in the bag:
- PGK (1)
- BGHpA (5)
- eGFP+k_GOI (6)
The fragments will be frozen and later purified.
We still have not succeed to make BioBricks pyrG and pyrG-DR (used as a marker in fungi), despite different PCR attempts with a high annealing (59°) and low (56°) temperature.
Work for iGEM Copenhagen team
We have established collaboration with the iGEM team from Copenhagen University. We will costumize a number of their BioBricks to use with our Plug 'n' Play system. This is a great way of showing how easy it is to costumize the Plug 'n' Play system for any application, and the Copenhagen team will save a lot of time.
Today we transformed competent E.coli cells with some of the standard biological parts from iGEM, BBa_R0010, Bba_B0015 and BBa_J52034, and later we will make them fit the Plug 'n' Play with DNA system.
Friday
We made a PCR of a troublesome BioBrick again - but this time with a small modification. We made a PCR mix with and without MgCl2 50mM in the reactions. And...IT WORKED with MgCl2 50mM!
Biobrick obtained:
The fragments will be frozen and later purified .
Week 4: 25.07.2011 - 31.07.2011
Monday
We got a new delivery of primers from IDT today. They are to be diluted and stocked, before use.
We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.
The PCR reactions were successful, and following biobricks are ready for Plug'n'Play:
- SV40 +ori (2)
- SV40 pA (4)
- LacZ (8)
Tuesday
The primers recieved yesterday will be used for amplification of many many many new biobricks today.
Out of 15 PCR reactions 5 succeeded:
- GFP_GOI (23)
- GFP_modul (24)
- GFP_TS (25)
- eGFP_TS (31)
- eGFP_module (32)
Wednesday
Today we have run 8 reactions, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.
One succeeded:
August
Week 5: 01.08.2011 - 07.08.2011
Monday
New week, new energy, and lots of team spirit!
Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!
New biobricks in the bag:
- RFP_GOI (20)
- RFP_modul (21)
- RFP_TS (22)
- bleR (29)
- pgpdA (26)
- yA (27)
- Plasmid_fun (28)
- Plasmid_mam (30)
- Amp cas (C1)
- Hygromycin (18)
- CYP79A2 (Copenhagen part) (C4)
- Terminator (Copenhagen part) (C6)
- pIPTG (Copenhagen part) (C2)
- CYP79B2(Copenhagen part) (C5
Wednesday
There are still 2 BioBricks that we haven't been able to obtain with PCR: pyrG and pyrG-DR.
Today we tried again with gradient PCR to find the optimal melting temperature, but no magic happened.
Thursday
22 BioBricks, which have been obtained with PCR during the last week of laboratory work, was purified today. The purification of DNA was done by using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The procedure was carried out according to the manufactures protocol for purification of DNA from TAE and TBE agarose gels, see protocol section for more information.
Despite several attempts we have not been able to obtain BioBrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, and thereby it revealed that the DNA template was the source of error. Problem solved!
We also started cultivation of the mammalian cell line, U2OS, which is derived from a patient with osteosarcoma (bone cancer). They were defrosted and cultivated in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section.
Week 5: 08.08.2011 - 14.08.2011
Monday
Today we made the first USER cloning, we are very excited to see if the system works the way, we want it to!
All our biobricks are divided into devices, see protocol for USER MIX.
The following devices were cloned today:
- Device 1: plasmid_mam (30) + CMV (3) + eGFP (32) + Hgh pA (33) + Hygromycin (18)
- Device 2: plasmid_mam (30) + SV40+ori (2) + eGFP (32) + SV40 pA (4) + Hygromycin (18)
- Device 3: plasmid_mam (30) + PGK (1) + eGFP_module (32) + BGH pA (5) + Neomycin (19)
- Device 4: plasmid_fun (28) + pAlc (7) + LacZ (8) + T1 (14) + argB (17)
- Device 5: plasmid_fun (28) + gpdA (26) + GFP_module (24) + trpC (13) + hygR (12)
- Device 6: plasmid_fun (28) + gpdA (26) + RFP_module (21) + trpC (13) + hygR (12)
- Device 7: plasmid_mam (30) + pIPTG (C2) + CYP79B2 (C5) + Terminator (C6) + amp (C1)
- Device 8: plasmid_mam (30) + pIPTG (C2) + CYP79A2 (C4) + Terminator (C6) + amp (C1)
PCR reactions done today:
- eGFP+k_GOI (6)
- eGFP_TS (31)
- yA (27)
- GFP_TS (25)
- RFP_TS (22)
- RFP_GOI (20)
- MTS (34)
- GFP_PTS1_fun (35)
- GFP_PTS1_mam (36 )
- RFP_NLS_module (37)
- pyrG (10 )
- pyrG-DR (11)
After new DNA template we finally succeed in getting biobricks pyrG and pyrG-DR.
The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration.
Tuesday
We inoculated E.coli with the plasmides done by USER cloning yesterday.
PCR done today:
- RFP_TS (22)
- RFP_GOI (20)
- RFP_NLS_Module (37)
Purification with GFX kit on the following biobricks:
- eGFP+K_GOI (6)
- eGFP_TS (31)
- yA (27)
- GFP_TS (25)
- RFP_TS (22)
- MTS (34)
- GFP_PTS1_module_fun (35)
- GFP_PST1_module_mam (36)
- RFP_GOI (20)
- RFP_NLS_module (37)
- pyrG (10)
- pyrG-DR (11)
USER cloning done today:
- Device 9: plasmid_fun (28) + gpdA (26) + MTS (34) + GFP_TS (25) + TtrpC (13) + hygR (12)
- Device 10: plasmid_fun (28) + gpdA (26) + GFP_PTS1_module_fun (35) + TtrpC (13) + hygR (12)
- Device 11: plasmid_fun (28) + gpdA (26) + RFP_NLS_module (37) + TtrpC (13) + hygR (12)
- Device 12: plasmid_mam (30) + CMV (3) + GFP_PST1_module_mam (36) + BGH pA (5) + Hygromycin (18)
- Device 13: plasmid_mam (30) + CMV (3) + eGFP_module (32) + BGH pA (5) + Hygromycin (18)
Wednesday
The U2OS cells are checked out in the microscope. They had a confluency of about 90%, which means that they are ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")
Friday
Today we did 13 PCR reaction, some of them includes parts to characterise our two promoters pAlc and DMKP-P6 and some parts for iGEM Copenhagen team.
- GFP_GOI (23)
- GFP_PTS_fun (35)
- GFP_PTS_mam (36)
- RFP_NLS (37)
- DMKP_P6 (38)
- ptrA (40)
- 1A2 (C7)
- 2C9 (C8)
- pAlc-L (B1)
- DMKP_P6-L (B3)
- RFP_TS (22)
- RFP_GOI (20)
The U2OS cells are splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday.
Week 7: 15.08.2011 - 21.08.2011
Monday
The U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They are placed in the incubator overnight - and tomorrow they will be ready for transfection with plasmides.
Preparing for characterization of two promoters with β-galactosidase assay were the plasmid p68 cut with restriction enzyme.
Purification of PCR products done last Friday were done with GFX kit.
9 PCR reactions were done today
- eYFP_module (41)
- eYFP_GOI (42)
- eYFP_TS (43)
- eCFP_module (47)
- eCFP_GOI (48)
- eCFP_TS (49)
- mCheryy_module (44)
- mCherry_GOI (45)
- mCHerry_TS (46)
USER cloning done today and transformed in E.coli today:
- Device 21: plasmid_fun (28) + PAlc (7) + LacZ (8) + T1 (14) + ptrA (40)
- Device 22: plasmid_fun (28) + gpdA (26) + GFP_module (24) + TtrpC (13) + pyrG (10)
- Device 23: plasmid_fun (28) + gpdA (26) + GFP_PTS1_module_fun (35) + TtrpC (13) + pyrG (10)
- Device 24: plasmid_fun (28) + gpdA (26) + RFP_module (21) + TtrpC (13) + pyrG (10)
- Device 25: plasmid_fun (28) + gpdA (26) + RFP_NLS_module (37) + TtrpC (13) + pyrG (10)
- Device 26: plasmid_fun (28) + gpdA (26) + MTS (34) + GFP_TS (25) + TtrpC (13) + pyrG (10)
- Device 28: plasmid_fun (28) + DMKP_P6 (38) + GFP_GOI (23) + RFP_TS (22) + T2 (15) + bleR (29)
- Device 29: plasmid_fun (28) + PAlc (7) + RFP_GOI (20) + RFP_TS (22) + T3 (16) + pyrG_DR (11)
- Device 13: plasmid_mam (30) + CMV (3) + eGFP_Module (32) + BGH pA (5) + Hygromycin (18)
- Device 12: plasmid_mam (30) + CMV (3) + GFP_PTS1_module_mam (36) + BGH pA (5) + Hygromysin (18)
- Device 17: plasmid_mam (30) + PGK (1) + eGFP+K_GOI (6) + eGFP_TS (31) + hGH_polyA (33) + Neomycin (19)
Tuesday
We transfected U2OS cells with plasmides containing GFP and placed them in the incubator.
USER cloning done today and transformed in E.coli today:
- Device 14: plasmid_mam (30) + CMV (3) + eYFP_module (41) + BGH pA (5) + Hygromysin (18)
- Device 15: plasmid_mam (30) + CMV (3) + mCherry_module (44) + BGH pA (5) + Hygromysin (18)
- Device 16: plasmid_mam (30) + CMV (3) + eCFP_module (47) + BGH pA (5) + Hygromysin (18)
- Device 18: plasmid_mam (30) + PGK (1) + eYFP_GOI (42) + eYFP_TS (43) + hGH_polyA (33) + Neomycin (19)
- Device 19: plasmid_mam (30) + SV40+ori (2) + mCherry_TS (45) + mCherry_TS (46) + SV30 pA (4) + Neomycin (19)
- Device 20: plasmid_mam (30) + Sv40+ori (2) + eCFP_GOI (48) + eCFP_TS (49) + SV40 pA (4) + Neomycin (19))
- Device 21: plasmid_fun (28) + PAlC (7) + LacZ (8) + T1 (14) + ptrA (40)
Wednesday
Inoculations of E.coli transformants with USER devices done the day before.
Restriction analysis of Device 4, 7, 8 and 27
USER cloning done today and transformed in E.coli today:
- Device 4: plasmid_fun (28) + PAlc (7) + LacZ (8) + T1 (14) + argB (17)
- Device 21: plasmid_fun (28) + PAlc (7) + LacZ (8) + T1 (14) + ptrA (40)
- Device 22: plasmid_fun (28) + gpdA (26) + GFP_module (24) + TtrpC (13) + pyrG (10)
- Device 23: plasmid_fun (28) + gpdA (26) + GFP_PTS1_module_fun (35) + TtrpC (13) + pyrG (10)
- Device 24: plasmid_fun (28) + gpdA (26) + RFP_module (21) + TtrpC (13) + pyrG (10)
- Device 25: plasmid_fun (28) + gpdA (26) + RFP_NLS_module (37) + TtrpC (13) + pyrG (10)
- Device 26: plasmid_fun (28) + gpdA (26) + MTS (34) + GFP_TS (25) + TtrpC (13) + pyrG (10)
- Device 28: plasmid_fun (28) + DMKP_P6 (38) + GFP_GOI (23) + RFP_TS (22) + T2 (15) + bleR (29)
- Device 29: plasmid_fun (28) + PAlc (7) + RFP_GOI (20) + RFP_TS (22) + T3 (16) + pyrG_DR (11)
- Device 31: plasmid_mam (30) + pIPTG (C2) + 1A2 (C7) + Terminator (C6) + amp_cas (C1)
- Device 32: plasmid_mam (30) + pIPTG (C2) + 2C9 (C8) + Terminator (C6) + amp_cas (C1))
- Device 7: plasmid_mam (30) + pIPTG (C2) + CYP79A2 (C5) + Terminator (C6) + amp_cas (C1)
- Device 8: plasmid_mam (30) + pIPTG (C2) + CYP79B2 (C4) + Terminator (C6) + amp_cas (C1)
Purifications of plasmid from transformed E.coli with mini-prep and restriction analysis of the plasmids.
Thursday
The coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.
Transformation of Aspergillus nidulans for promotor characterization.
Friday
USER cloning done today and transformed in E.coli today:
- Device 28: plasmid_fun (28) + DMKP_P6 (38) + GFP_GOI (23) + RFP_TS (22) + T2 (15) + bleR (29)
- Device 22: plasmid_fun (28) + pgdA (26) + GFP_module (24) + TtrpC (13) + pyrG (10)
- Device 30: plasmid_fun (28) + PAlc (7) + yA (27) + RFP_TS (22) + TtrpC (13) + amp_cas (C1)
Week 8: 22.08.2011 - 28.08.2011
Monday
The U2OS cells are splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.
Tuesday
The U2OS cells are transfected with all the mammalian plasmides. So hopefully we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.
Measurements of plasmids with our device for sequencing. The measurement were done with Qubit Fluorometer from Invitrogen.
And more restriction analysis of plasmids were done.
Wednesday
The coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great - everything worked = we are done with the mammalian cells. Hip hip hurra!
Transformation of Aspergillus nidulans for proof of concept.
September
Week 9: 29.08.2011 - 04.09.2011
Tuesday
All plasmids and primeres were sent to sequencing.
Wednesday
PCR reaction of the shipping plasmid were done. The shipping plasmid had to modified with our USER tails so we quickly could clone our 2 biobricks and one device to send.
Thursday
USER cloning of the two promoters and device 12 with the shipping plasmid were done.
Due to the extensive use of biobrick plasmid_fun(28) and plasmid_mam(30) we did a PCR reaction again so it can be sent to iGEM.
Week 10: 05.09.2011 - 11.09.2011
Monday
Restriction analysis of shipping plasmid, to check if the USER cloning were done correct. Shipping plasmid were sent to sequencing.
Inoculation of fungi for β-galactosidase and Bradford assay Wednesday and Fluorescence microscope Tuesday.
Tuesday
Today we looked at our fungi in fluorescence microscope, and what a beautiful sight it was! We have now prooven our system works in fungi as well as mammalian cells.
Wednesday
Today we spent all day in the lab with β-galactosidase and Bradford assay for charactisation of our two promoters.
Friday
Hopefully this day will be one of the last in lab. We are measuring the DNA concentration of our biobricks so they can be sent to MIT next week.
October
November
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