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Team:UANL Mty-Mexico/Notebook
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| - | <figcaption><span class="text-muted"><br>Figure 4. Electrophoresis gel from the August 30 miniPreps</span> <br> | + | <figcaption><span class="text-muted"><br>Figure 4. Electrophoresis gel from the August 30 miniPreps cut with <i>Hin</i>fI, Lambda-<i>Pst</i>I as marker.</span> <br> |
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Latest revision as of 03:00, 29 October 2013
Notebook
Reference Data
| Part Name | Part Short name | In pSB 1A3 (bp) | In pSB 1C3 (bp) | In pUC57 (bp) | Part size (bp) |
|---|---|---|---|---|---|
| RBS + TetR | TetR | 2,925 | 2,802 | 3,478 | 732 |
| pLac + GFP | GFP | 3,162 | 3,034 | 3,715 | 964 |
| pCons LacI | LacI | 3,483 | 3,352 | 4,036 | 1,282 |
| pTetR + mCherry | mCherry | 3,107 | 2,957 | 3,660 | 887 |
| BBa_K1140005 | 3-E1 | 2,811 | --- | --- | 935 |
This table works as a reference of the parts that were used in the laboratory and in the diary. We have a short name for each part so for example, if a diary entry says "TetR" we mean the complete part "RBS + TetR". Another important aspect is that we were working with the plasmids pSB 1C3, pSB 1A3 and pUC57 so here there is the information about the length of the part and the length in each plasmid.
August 4th, 2013
Today we prepared solutions in order to start working on the lab the following day. The solutions will be required for the MiniPrep, Transformation. We also prepared aliquots, mQ water with RNAse, etc.
August 5th, 2013
Today we started with the transformations of the synthetic DNAs that just arrived.
August 6th, 2013
We inoculated the colonies obtained from the transformations.
August 7th, 2013
This day we did Minipreparation of DNA. The parts that were obtained were the following: LacI, GFP, mCherry, TetR and 3-1E
August 8th, 2013
Test of mCherry with Thermo-mixer-Cualitative experiment
20 Colonies of the dish with pTet-R-mCherry were planted in 0.5 mL of Agar with Kanamycin in order to observe the red colour after several hours at 42ᵒC. The experiment was performed following these parameters.
| Start | End | Hours | |
|---|---|---|---|
| 37ºC | 2:50 pm | 7:02 pm | 4 hr 12 min |
| 42ºC | 7:05 pm | 10:35 am | 15 hr 40 min |
Most tubes shown development seeing them on the light but there was no color shown.
August 10th, 2013
20 tubes with mCherry were put in the shaker with 2 controls with CDS (with oxygen everyone), following these parameters:- Temperature: 37ᵒC
- Revolutions: 900 rpm
- Start: 11:35 am -> 9:00 am (Time 21:35 hours)
The tubes show mCherry expression, some more intense than others. The experiment was repeated at 30°C overnight with a volume of 500 µL at 30°C with tubes of 1.5 ml. They were left at 4°C meanwhile they were incubated.
| Set | Temperature | Velocity | Time | Live |
|---|---|---|---|---|
| Black Set | 42ºC | 900 rpm | 15 hours | :( |
| Blue Set | 37ºC | 900 rpm | 21 hours | :) |
| Red Set | 32ºC | 900 rpm | :) |
Time 10:25 – 9:15 = 22:50 hrs
August 12th, 2013
The experiment was repeated at 42°C allowing the oxygen in to the tube. It began at 2:15 pm and ended at 1:20 pm of the next day.
Figure 1. Tubes with the mCherry pellet that were incubated at 42ºC.
August 16th, 2013
Electrophoresis gel of the digestions from 15/08/13
Figure 2. Electrophoresis gel of the digestions from 15/08/13
| DNA | 1x | 5x |
|---|---|---|
| DNA | 2 µL | |
| EcoRI | 0.3 µL | 1.5 µL |
| PstI | 0.3 µL | 1.5 µL |
| Buffer O (Fermentas) | 1 µL | 5 µL |
| 10 µL | 50 µL |
August 18th, 2013
There where no colonies present from the previous day.
Ligation with a 1:1 ratio DNA:Vector. Is was performed with the following parameters:
| Size (bp) | Vector | DNA:Vector | |
|---|---|---|---|
| pTetR | 768 | 2.8 | 0.33/1 |
| pCons-LacI | 1326 | 1.6 | 0.62/1 |
| pTetR-mCherry | 960 | 2.2 | 0.45/1 |
| pLacI-GFP | 1005 | 2.1 | 0.47/1 |
August 19th, 2013
This ligation was not successful in the transformation.
Dishes of:pTet-mCherry, pCons-LacI, pTetR, pLacI-GFP, and the 3-1E DNA were planted again.
August 26th, 2013
All the dishes were transformed but only the pLacGFP grew successfully.
August 28th, 2013
Today we picked up colonies from the dishes from the previous day. We also prepared new competent cells.
August 29th, 2013
All the dishes were successfully transformed and we planted in petri dishes. We got colonies of all the parts, they grew in the test tubes and miniPrep was done.
August 30th, 2013
The MiniPrep was successful and we did digestions with EcoRI. Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP. *They were planted in Ampicilin dish.
Figure 3. Electrophoresis gel from the August 30 miniPreps
August 31th, 2013
MiniPrep and digestions from the ones of 08/30/2013. We didn't obtained any construction.
Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP
*They were planted in Ampicilin dish.
September 11th, 2013
Digestions with EcoRI and PstI and Ligations
September 12th, 2013
We didn't obtained any part so we started again transforming them one more time.
September 13th, 2013
There were no results presented so we transformed again.
September 14th, 2013
We got no results in the construction desired.
September 19th, 2013
Quantification with the Nanodrop of the previous samples
| ng/µL | 260/280 | |
|---|---|---|
| pSB1C3 | 1293.4 | 1.99 |
| GFP | 133.5 | 1.84 |
| TetR | 119.6 | 1.78 |
| tRFP | 104.3 | 1.74 |
| pConsLac | 130.5 | 1.59 |
September 20th, 2013
We prepared new calcium competent cells, E. coli strain Top10.
September 23th, 2013
We did the transformations again for all the parts.
September 24th, 2013
Again there where no results.
Note: The constructions with the pSB1A3 and pSB1C3 showed no results but the controls with pUC vector were fine and did work in our experiments.
October 9th, 2013
New Digestions with EcoRI and PstI.
October 10th, 2013
New ligations were done
October 11th, 2013
New Petri dishes were made, and use for transformation today.
October 13th, 2013
We inoculated the colonies obtained from the transformations.
October 14th, 2013
We did minipreparation of DNA. After, the DNA was digested with EcoRI, PstI and HinfI for an enzymatic analysis (our Gel #700!!!). After hours, we did a electrophoresis and we realised that 2 new parts were now in the pSB 1C3 vector.
Figure 4. Electrophoresis gel from the August 30 miniPreps cut with HinfI, Lambda-PstI as marker.
October 17th, 2013
We started a new test in order to measure number of plasmids per cell in M1, M2 and M12.
October 18th, 2013
First results for number of plasmids.
October 21th, 2013
More results for number of plasmids.
October 22th, 2013
A test for measuring expression of GFP (in pUC57 and pSB1C3) was prepared in microcentrifuge tubes with LB medium and IPTG 1mM for 17 hours at 37 °C (in bacteria E. coli Top10) in thermomixer.
We still have problems with expression of mCherry, so we did new cultures with stored (red) M1, M2 and M12 in order.
October 23th, 2013
We used fluorometer to measure expression of GFP, but any fluorescence was detected, so new microcentrifuge tubes with GFP construction were prepared at same conditions of last day.
The cultures of M1, M2, M11 and M12 were red and presented fluorescence, so we did minipreparation and culture again them in new tubes. With the DNA that was obtained, a digestion with EcoRI and PstI was done and a new ligation.
October 24th, 2013
Again, any fluorescence was detected for GFP cultures. New microcentrifuge tubes with GFP construction were prepared with IPTG 0.5 mM.
A new transformation was done, using new mCherry and latest TetR and LacI ligations.
Cultures of M11 and M12 grew, but any with M1 and M2. We did minipreparation and the DNA was measured in nanodrop.
October 25th, 2013
Residual fluorescence was detected for GFP cultures.
We inoculated the colonies obtained from the transformations.
October 26th, 2013
Cultures of mCherry grew, but the others did not. We did minipreparation of that tubes and digested them with EcoRI, PstI and HinfI. The results can be seen on gel # 709 (EcoRI/PstI) and 710 (HinfI). Probably, we have multiple clones with mCherry in pSB1C3.
New ligations of TetR, LacI and GFP with pSB1C3 were done and a transformation was made in a new competent stock, Top10 strain.
Figure 5. Electrophoresis gel (709) with the EcoRI/PstI clones digestion, Lambda-PstI as marker.
Figure 6. Electrophoresis gel (710) with the HinfI clones digestion, Lambda-PstI as marker.
October 28th, 2013
We did minipreparation and digested them with HinfI to identify more positive clones. As a result we get at the most two positive clones, one for pSB-mCherry and one for pSB-GFP
Figure 7. Electrophoresis gel with the HinfI clones digestion for mCherry, GFP, LacI TetR with pSB1C3 vector as control, Lambda-PstI as marker.
Stability
For the stability experiments the tests were performed from September 14 to 20. The protocols are in the section of Safety. Click here
For the fluorescence experiments the tests were performed from September 10 to 27. The protocols are in the section of Wetlab. Click here
