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Team:NTNU-Trondheim/Notebook/July
From 2013.igem.org
We transformed ''E.coli'' strain DH5α with some BioBricks: pBAD promoter (BBa_K206000), RBS (BBa_J61101) and a plasmid backbone (BBa_J01101).
This day we started making the small-scale vesicle preparation according to our protocol.
Overnight cell cultures of ''E.coli'' ER2566 and ''E.coli'' BW27784 transformed with the pUM9 plasmid were preprared. The pUM9 plasmid has ampR induces a stress respons in ''E.coli'' when arabinose is present. According to the litterature (link?) stress in ''E.coli'' increases vesicle production.
The ER2566 cell were cultured in plain LB, while the BW27784 cells were cultured in LB with ampenicillin (100 µg/mL).
We transferred the samples of tranformed DH5α (pBAD promoter (BBa_K206000), RBS (BBa_J61101) and a plasmid backbone (BBa_J01101) from agar plate to liquid medium and incubated at 37°C overnight.
The small-scale vesicle preparation continued with completion of step 2-7 in the vesicle isolation protocol. There where made three cell cultures in step 2; ''E.coli'' ER2566 in LB, ''E.coli'' BW27784 in LB with ampenicillin (100 µg/mL) (ara-) and ''E.coli'' BW27784 in LB with ampenicillin (100 µg/mL) with the addition of arabinose (0.5 %) after 1 hour of incubtion (ara+). The cell cultures in step 2 was incubated for 3 hours.
Optical denisty (OD) at 600 nm was mesured as indicated in the table below. No dilution of the samples was necessary.
| Sample | OD600 |
|---|---|
| ER2566 | 0.9805 |
| ara- | 0.1263 |
| ara+ | 0.0471 |
We isolated the DNA from the transformed cells pBAD promoter (BBa_K206000), RBS (BBa_J61101) and a plasmid backbone (BBa_J01101) by using the miniprep protocol and measured the concentration by NanoDrop ND-1000 Spectrophotometer.
| Sample | Concentration |
|---|---|
| RBS | 4,91 |
| pTet1 | 8,99 |
| pTet2 | 4,39 |
| pBAD1 | 3,54 |
| pBAD2 | 9,24 |
Continuation of the small-scale vesicle preparation according to the vesicle isolation protocol. Step 8-12 was completed with the exception that the pellet was resuspended in 0.5 mL DPBSS insted of 100 µL in step 11 and that the check for sterility in step 12 was not performed. The SDS-PAGE showed no signs of vesicles as viewed in figure 1.
Figure 1.
An overnight culture was started for vesicle isolation as desribed for tuesday 02.07.10.
Today we ran PCR on 4 FP's BBa_K864100, BBa_K592100, BBa_E0040, E1010 and 1 backbone BBa_J01101. We used the Phusion DNA Polymerase protocol from New England Biolabs (NEB) for 50 ul sample(without DMSO). All amplicons were run on a GelGreen 0.8% agarose gel. We only got a band on our RFP E1010, and it was not the expected size. We will adjust the PCR parameters and run them again tomorrow.
Step 2-11 of the small scale-vesicle preparation protocol and step 1-4 of the density gradient protocol was performed. Samples from the small-scale vesicle preparation were made ready for SDS-PAGE.
The incubation period was revised from the first attempt to isolate vesicles. The duration and other condition is summerised in the table below.
| Sample | Sample | Sample | Time (h:min | ER2566 | MW27784 ara- | MW27784 ara+ |
|---|---|---|---|
| 0:00 | Add 250 μL Amp (100X) and 2,5 mL of overnight culture to 250 mL of LB. Incubate at 37 C | Add 250 μL Amp (100X) and 2,5 mL of overnight culture to 250 mL of LB. Incubate at 37 C | |
| 1:00 | Add 1 mL of overnight culture to 250 mL of LB. Incubate at 37 C | ||
| 2:00 | Add 1,25 μL arabinose (1000X) | ||
| 4:00 | Measure OD-600 | Measure OD-600 | Measure OD-600 |
Optical denisty (OD) at 600 nm was mesured in step 4 as indicated in the table below. No dilution of the samples was necessary.
| Sample | OD600 |
|---|---|
| ER2566 | 0.1671 |
| ara- | 0.6726 |
| ara+ | 0.5968 |
We ran the PCR again with different parameters. Increased annealing- and elongationtime and a separate program for the backbone due to its larger size. We still only got a band for RFP E1010. In the prosess of rechecking the primers it was discovered that the primers for RFP are incorrect which explains the discrepancy in productsize. The other primers should be working so tomorrow we will redo the dilutions of the primers, increase the elongation time and lower the annealing temperature a little, add DMSO, add a bit larger templatesample and use 1-step PCR.
SDS-PAGE were run with the samples from the vesicle preparaton from the day before. There were no indication of vesicles in the samples as indicated on the gel (figure 2).
Figure 2.
Today we ran PCR again. This time we omitted the RFP, since we discovered that one of the primers was incorrect. We diluted the primers one more time and changed the parameters for the PCR. This time we only did one step, with 30 cycles. We also decreased the annealing temperature and increased the elongationtime. The results were more uplifting this time around after running gel electrophoreses (picture coming soon). We got a clear band on the backbone and faint bands on the GFP and SYFP. Since there was a faint band on the backbone of a larger size than the one we wanted, we added 1 µl of Dpn1 to the amplicon and put it in a 37 °C waterbath for an hour. We used the QIAGEN PCR purification kit to isolate the DNA and measured the cioncentration by NanoDrop ND-1000 Spectrophotometer.
| Sample | Concentration (ng/µl) |
|---|---|
| SYFP | 13,16 |
| GFP | 55,62 |
| BB | 55,50 |
We repeated the PCR on the SYFP and BFP. We changed some of the conditions (increased number of cycles and varying the template volume), but still no result.
Step 1 and 2 of the [https://2013.igem.org/Team:NTNU-Trondheim/Protocols Small-scale vesicle preparation] were performed. 5 mL cultures of the ''E.coli'' strains ER2566, DH5α and MW27784 were incubated in 5 mL LB at 37 oC for 8 h. 2,5 mL of these cell cultures were added to 250 mL of LB and incubated at 37 oC for 13 h.
Step 3-11 of small scale-vesicle preparation protocol and step 1-4 of the density gradient protoco was performed. Samples from the small-scale vesicle preparation were made ready for SDS-PAGE.
Optical denisty (OD) at 600 nm was mesured in step 4 as indicated in the table below. The samples were diluted 1:16 with LB media.
| Sample | OD600 |
|---|---|
| ER2566 | 0.133 |
| DH5&alpha | 0.169 |
| MW27784 | 0.198 |
A relative concentration of vesicles from the small scale preparation was mesured by adding the hydrophobic fluorecent dye FM4-64 and measuring fluorecence (RFU) (as desribed in step 12 in the small-scale vesicle preparation). Results (table below) indicated presence of vesicles in the ER2566-sample only.
| Sample | RFU (exitation/emission at 506/750 nm) |
|---|---|
| Empty | 3 |
| Blank | 19814 |
| MW27784 | 19598 |
| DH5α | 19 254 |
| ER2566 | 40 987 |
Two rounds of PCR was run today with varying the conditions.There are still no result. For the BFP, we will wait for new primers, since the result are showing formation of primer dimers. A new transformation of the SYFP-biobrick was started to get a new template for the PCRs.
SDS-PAGE were run with the samples from the vesicle preparaton from the day before. The ER2566-sample had vesicles, where as the other samples did not contain any (figure 3).
Figure 3: Ladder applied is Precision Plus ProteinTM Unstained Standards..
Step 5 of the purification of vesicles by density gradient was performed. SDS-PAGE were run on the fractions from ER2566, as this was the only sample with any indication of vesicles. As indicated in figure 4, fraction 4 and 5 had clear bands, whereas the fraction 6 has pale bands. The fractions are numbered according to when whey where removed from the gradient with fraction 1 beeing the first fraction to be removed and so on.
Figure 34: Ladder applied is Precision Plus ProteinTM Unstained Standards.
The rest of the fractions were freezed down at -80 oC. Too short incubation time in step 2 of the small-scale vesicle preparation seem to be the reason why vesicles were not isolated during the two previous attempts.
Due to postive results, the Small-Scale vesicle preparation was repeated: Step 1 and 2 of the Small-Scale vesicle preparation was performed. 5 mL cultures of the ''E.coli'' strains ER2566, DH5α and MW27784 were incubated in 5 mL LB at 37 oC for 8 h. 2.5 mL of these cell cultures were added to 250 mL of LB and incubated at 37 oC for 14 h.
Step 3-11 of the Small-Scale vesicle preparation protocol was performed on the ER2566, DH5α, and MW27784-samples.
Optical denisty (OD) at 600 nm was mesured in step 4 as indicated in the table below. The samples were diluted 1:10 with LB media.
| Sample | OD600 |
|---|---|
| ER2566 | 0.281 |
| DH5&alpha | 0.293 |
| MW27784 | 0.402 |
The DH5α had no pallet after the second centrifugation, and this sample where therefore not included further in the experiement. Some of MW27784 sample was lost in step 10 of the small-scale vesicle preparation. A part of the ER2566 and MW27784-samples from the small-scale vesicle preparation were made ready for SDS-PAGE. The rest of the samples were mixed with OptiPrep (60%) to give a final OptiPrep concentration of 45% and frozen at -80oC.
A relative concentration of vesicles from the small scale preparation was mesured by adding the hydrophobic fluorecent dye FM4-64 and measuring RFU (as desribed in step 12 in the small-scale vesicle preparation). This time we also measured RFU with exitation at 515 nm and emission at 640 nm. As the last exitation and emission gave a more significant difference between the blank sample and the vesicle containing sample, this condition will be applied for later RFU-measurements. Results (table below) indicated presence of vesicles in the ER2566-sample and perhaps also in the MW27784-sample.
| Sample | RFU (exitation/emission at 506/750 nm) | RFU (exitation/emission at 515/640 nm) |
|---|---|---|
| Empty | 1 | 32 |
| Blank | 16536 | 1180 |
| MW27784 | 16335 | 1691 |
| ER2566 | 47 191 | 44 059 |
We isolated the DNA from the transformed cells (with SYFP) by using the miniprep protocol and measured the concentration by NanoDrop ND-1000 Spectrophotometer.
| Sample | Concentration (ng/µl) |
|---|---|
| SYFP1 | 30,5 |
| SYFP2 | 20,8 |
We used the new isolated DNA as template in PCR, trying different parameters.
SDS-PAGE were run with the samples from the vesicle preparaton from the day before. The ER2566-sample had a clear content of vesicles whereas the MW27784-sample had a less denser content of vesicles (figure 5). The MW27784 also seems to have a different protein profile.
Figure 5: Ladder applied is Precision Plus ProteinTM Unstained Standards.
''E.coli'' strain ER2566 was transformed with biobrick BBa_K530015 according to the transformation protocol. The plates were incubated for 19 hours and then moved to the fridge. The transformed ER2566 bacteria is later to be used for determination of influence of chloramphenicol on the cells and for growth duration for optimal vesicle production.
Three cell cultures of ''E.coli'' strain ER2566 transformed with BBa_K530015 was inoculated in Chl-LB and one cell culture of MW27784 were inoculated in LB for 7.5 hours at 37°C as described in step 1 of the small-scale vesicle preparation. The transformed ER2566 seems to grow slower in Chl_LB than untransformed ER2566 does in plain LB. Bigger overnight cell cultures were incubated as in step 2 of the small-scale vesicle preparation, the ER2566 cells were incolated in Chl-LB as before. The three ER2566 samples were incubated for 14, 16 and 18 hours, while the MW27784 sample was incubated for 16 hours.
Step 4-12 of the small-scale vesicle preparation were performed. In addition dilutions (10-2, 10-4 and 10-6) of the ER2566 samples were made and plating of 100µL of 1 mL of the dilutions on Chl-LA was performed in step 4. The plates were left in the incubator overnight.
Optical denisty (OD) at 600 nm was mesured in step 4 as indicated in the table below. The samples were diluted 1:10 with LB media.
| Sample | OD600 |
|---|---|
| ER2566 14h | 0.247 |
| ER2566 16h | 0.347 |
| MW27784 16h | 0.533 |
| ER2566 18h | 0.328 |
Samples for SDS-PAGE was prepared in step 12.
RFU was measured as decribed in step 12 of the small-scale vesicle preparation protcol. Results were as indicated in the table below:
| Sample | RFU (exitation/emission at 515/640 nm) |
|---|---|
| Empty | 91 |
| Blank | 6 745 |
| ER2566 14h | 31 571 |
| ER2566 16h | 45 089 |
| MW27784 16h | 6 899 |
| ER2566 18h | 22 328 |
Plates with transformed ER2566, plated the day before, were checked for colonies. As the 10-2 and 10-4 dilution had to much growth, only the 10-6-dilutions were counted for colony forming units (CFU)(table below):
| Sample | CFU (10-6-dilution) | CFU/mL (undiluted |
|---|---|---|
| ER2566 14h | 265 | 2.65*109 |
| ER2566 16h | 278 | 2.78*109 |
| ER2566 18h | 273 | 2.73*109 |
| ER2566 | 47 191 | 44 059 |
It seems that transformed ER2566 reaches steady state at 14h or quickly after 14h in the 240 mL cellmedia. The CFU results corresponds well with the measured OD600 (table from the day before) except for that the OD600 of ER2566 14h is more different from the other samples compared to the CFU-results.
SDS-PAGE were run on the samples prepared the day before. There were very weak bands on the on all of the ER2566 samples (not as visible in the photograph (figure 6) as in real life) and no indication of vesicles on the MW27784-sample.
Figure 6: Ladder applied is Precision Plus ProteinTM Unstained Standards.
The results of the SDS-PAGE does not correspond well to the results of the RFU-measurements from the day before. The RFU results indicate that the concentration of vesicles is higher.
We recieved the new primers for the GFP, RFP and BB(Backbone) that we intend to attach to each other with Gibson Assembly. We got results from all and purified DNA using NEB Purification Kit.
Since we cannot seem to get the primers for SYFP and BFP to work and they are both developed by the same iGEM team we're testing them for futher characterization. Transformation of SYFP, BFP and an appropritate BB with promoter and RBS were done and plated out. The plates are incubated in 37 degrees overnight and put in the frige.
Because the SDS-PAGE from yesterday did not give us expected results in terms of vesicles consentration, we started with the density gradient vesicle isolation with the ER2566 14h and 16h-samples to further test them for vesicles. Step 1-4 of this protocol was performed.
We isolated the tat-signaling sequence beloning to the TorA gene from the "E. Coli" strain ER2566 using our designed primers with PCR.
Step 5 and 6 of the density gradient vesicle isolation protocol was performed. The SDS-PAGE of the fractions show no or little indication of vesicles, further confirming the tendencies from the day before.
The Tat-PCR product was purified using NEB DNA Purification Kit. We will use our purified PCR products of Tat, GFP, RFP and BB in Gibson assembly.
The Gibson assembly reaction of the plasmid backbone, tat signal-sequence, GFP and RFP to create our first construct was performed according to the Gibson AssemblyTM Cloning Kit Manual. We made two samples, one with a incubation time (with the mastermix) of 60 min and one with 15 min. The control-sample was incubated for 15 min. The samples were then used for transformation of both ''E.coli'' strain DH5α and ER2566, giving us 4 samples. The transformation protocol was also collected from the TM Cloning Kit Manual. Transformed bacteria were incubated on plates with the appropriate antibiotics for 18 hours and then put in the fridge.
| Insert(I)/Vector(V) | Concentration(ng/µl) | Size (bp) | µl |
|---|---|---|---|
| Tat(I)5X | 35.84 | 150 | 1.5 |
| GFP(I)2X | 52.68 | 750 | 1.0 |
| RFP(I)2X | 98.31 | 750 | 1.0 |
| BB(V) | 73.82 | 2500 | 1.4 |
Add 10 µl of Gibson Mastermix and up to 20 µl dH2O.
There were no colonies on the plates where bacteria transformed with the 60 minutes incubation time Gibson assembly plasmids. There were three colonies with ER2566 transformed with the 15 minutes incubation time Gibson assenmbly plasmids and one colony on the plate with DH5α.
The colonies with SYFP, BFP and BB was transferred to liquid media.
Due to our unexpected vesicle yeild last week (see figure 6), we started a new round of small-scale vesicle preparation. Step 1 and 2 of this protocol was performed with ''E.coli'' strains DH5α, untransformed ER2566 and ER2566 transformed with biobrick BBa_K530015. The transformed ER2566 was inculated in LB with chloramphenicole. All cultures were incubated for 16 hours.
Isolation of SYFP, BFP and BB from last weeks transformation.
The GFP, BFP an BB wad digested using restrictionenzymes. We followed the protocol Restricion Digest. BB was digested with SpeI and PstI and SYFP/BFP with XbaI and PstI. The following ligation was performed using this Ligation Protocol using bot 1:1 and 1:2 vector insert ratio.
Step 4-12 of the small-scale vesicle preparation were performed. In addition a 10-6 dilution of the ER2566 samples were made and plating of 100µL of 1 mL of the dilution on Chl-LA was performed in step 4. The plates were left in the incubator overnight. The plate had growth, indicating that the bacteria still had their plasmid.
Optical denisty (OD) at 600 nm was mesured in step 4 as indicated in the table below. The samples were diluted 1:10 with LB media.
| Sample | OD600 |
|---|---|
| Unstransformed ER256614h | |
| Transformed ER2566 16h | |
| Unstransformed DH5α |
Samples for SDS-PAGE was prepared in step 12.
RFU was measured as decribed in step 12 of the small-scale vesicle preparation protcol. Results were as indicated in the table below:
| Sample | RFU (exitation/emission at 515/640 nm) |
|---|---|
| Empty | 93 |
| Blank | 22340 |
| Transformed ER2566 | 27249 |
| Untransformed ER2566 | 48478 |
| Unstransformed DH5α | 22029 |
From now on we will consentrate on ''E.coli'' strain ER2566.
Miniprep of Gibson Assembly construct and restriction digest gave wrong product on the gel. The size is not correct, so we must try again.
Analysis of Gibson Assembly construct to ensure that there is no contamination in our 4 construct component DNA we put 1µl of each DNA sample in 50µl of competent cells(ER2566) and plated them out. Incubated at 37°C overnight.
Analysis of Gibson Assembly constructWe used the same protocol, but changed the DNA concentration to 200 ng and increased the ratio of tat to 10X instead of 5X.
| Insert(I)/Vector(V) | Concentration(ng/µl) | Size (bp) | µl |
|---|---|---|---|
| Tat(I)5X | 35.84 | 150 | 2.5 |
| GFP(I)2X | 52.68 | 750 | 3.0 |
| RFP(I)2X | 98.31 | 750 | 1.5 |
| BB(V) | 73.82 | 2500 | 3.0 |
Add 10 µl Gibson Mastermix.
They where then transformed into ER2566 cells on 4 plates. Three plates with 5µl template, and one with standard 2µl template. Incubated at 37°C overnight.
| Plate | Templatevolume(µl) | Colonies |
|---|---|---|
| 1 | 2 | 3 |
| 2 | 5 | 36 |
| 3 | 5 | 37 |
| 4 | 5 | 250 |
Colonies where picked from all four plates and transferred to liquid media. Left overnight in 37 degrees shaker.
SDS-PAGE were run on the samples from the day before. There were only a few weak bands on the gel (not visible on a potential picture) indicating vesicles in the unstransformed ER2566-sample only.
A new round of small-scale vesicle preparation was started with two transformed ER2566 samples (same conditions) and one sample of unstransformed ER2566. The transformed ER2566 had a plasmid that wes a product from the first Gibson Assenbly. Step 1 and 2 of the protocol was completed with the transformed ER2566 incubating in LB with chloramphenicol (25µL/mL) and untransformed ER2566 incubating in plain LB.
We followed the procedure Restriction Digest in the registry.
| Template | Concentration(ng/µl) | µl | dH2O |
|---|---|---|---|
| 1 | 17.9 | 14 | 2 |
| 2 | 137 | 2 | 14 |
| 3 | 26.7 | 9.6 | 6.5 |
| 4 | 20.6 | 12 | 4 |
Incubated at 50 °ree;C for 15 min. Run on gel, positive result for sample 1. Will do confirmation PCR tomorrow.
