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May, 23th, 2013
Today we started officially the work in the lab. We resuspended all the biobricks we were going to use and transformed them. We also transformed a positive control at 1ng/µL that was not a biobrick.
May, 24th, 2013
Today we checked the Petri dishes and the positive control worked but the biobricks did not grow.
We quantified the DNA with the nanodrop but the results obtained were not reliable.
May, 29th, 2013
Today we made competent cells and we transformed 4 DNAs that are not biobricks and 2 biobricks that are not in our construction plan.
May, 30th, 2013
The competent cells worked very well, the 4 DNAs grew with an efficiency of 1.27x10^6 of the cells. The other 2 biobricks did not grow again. We tried again transforming the biobricks with the new cells.
May, 31th, 2013
Today we got cells of the GFP, YFP and CFP with degradation tag. We observed the cells in the microscope, the GFP with tag degradation seemed to be better than the YFP and the RFP. Also, today we transformed the plasmids pSB1T3, pSB1C3 and pSB1K3 that were sent to us accompanied by the biobricks. We picked up the colonies of the reporters and we placed them in a test tube with LB medium.
Image 1. Bacteria with GFP under the microscope.
June, 1st, 2013
We only got the plasmids pSB1K3 and pSB1C3. We picked up the colonies of both plasmids and we placed them in a test tube with LB medium.
June 3rd, 2013
The bacteria grew:
Image 2. Finally we have bacteria in tubes. We are ready to do our first miniPrep
We did MiniPreparation of Plasmidic DNA of the 3 reporters GFP, YFP, CFP and the 2 plasmids pSB1K3 and pSB1C3. We ran the electrophoresis gel with the 5 DNAs.
Image 3. Biobricks are ready after the mini Prep.
June 4th, 2013
We transformed the synthetic DNAs that just arrived today that are in the PCCI and PUC-57 plasmids. Also this day, we pick up some colonies from the samples of the 5-14O, 5-4E and 5-14I dishes; which were the same reporters that the ones used in the MiniPrep, but these samples were from previous samples then were from the past iGem team. These were stored in the incubator.
Image 4. Synthetic DNA's arrived today.
June 5th, 2013
We got a lot of colonies of PCCI and very few of the PUC-57. As the colonies with the cI biobrick did not grow, we transformed it again and a little more of the PUC-57. We also cloned cells to make competent cells.This day we noticed that the colonies of reporters that we picked up the day before didn’t grow. Again this day the colonies of GFP, CFP and YFP and also the PCC1 and PUC-57 were picked up and inserted in other tubes with other LB medium, and again were stored in the incubator.
Image 5. Colonies of the genes that just arrived yesterday.
June 6th, 2013
We are not able to make competent cells because the cells did not grow. No one of the colonies grew. We cloned again from the Petri dishes of the PCCI, PUC-57 and we tried again with the cI repressor biobrick, into the test tubes with LB medium.With the MiniPrep DNA’s done two days ago, a characterization was done in order to finally obtain the pieces and so we can be able to bind the genes. The reporters were cut, but when electrophoresed in agarose gel doesn’t show a good result.
June 7th, 2013
We got bacteria in the test tubes so we made miniPreparation of plasmidic DNA. We also cloned from tube to tube in order to have more cells as a backup. This day, the characterization was done again, now with more specifications with the enzymes. This time, the cuts went well with the 5-14I and 5-14G samples, because the image of the agarose gel showed the correct size. Also this day, MiniPrep was realized to the samples of the PCCI, PUC and 5-4E. The result of the electrophoresis showed a very degradated DNA, but still it showed a good result.
Image 6. Gel with the MiniPreps June 6th, 2013
June 8th, 2013
We did miniPreparation of DNA again.This day, the electrophoresis of yesterday MiniPreps was done again and it showed, as expected, more degradation of the DNA. Anyway, it was decided to do the cuts of the parts, each part with the respective enzymes:
- GFP was cut with EcoI and SpeI
- 5-4E was cut with XbaI and Pst
- PUCI was cut with EcoI and SpeI
After the electrophoresis, the results doesn’t showed a good cut and also showed the DNA very degraded.
Image 7. Gel with the MiniPreps June 6th degradated
June 10th, 2013
We prepared a digestion with the restriction enzymes with all the parts that we were going to need. This day it was done a replanting of the reporters GFP, CFP and YFP, and the plasmids 1-5A and 1-3A into LB medium and they were stored in the incubator. Also an electrophoresis was done with Friday’s cuts again, showing the same results.
June 11th, 2013
On this day, the colonies that were replanted yesterday doesn’t grow up, but even this we tried a MiniPrep in each sample. The results of the electrophoresis showed that only 3 DNA’s went well, while the other ones showed a faint DNA. Anyway, we decided to do the cuts with the enzymes of the 3 DNA’s that went well. Also this day, another replanting was done with the same samples of yesterday, to see if they grow correctly tomorrow. We ran a gel with only PCC1 and these were the results:
Image 8. Gel with only PCC1. First time it worked.
June 12th, 2013
The cuts that were done yesterday, were subject of an electrophoresis in agarose gel, the cuts showed the expected results except for the PCC1. It was decided to start all the MiniPreps again, to have a backup in case ligation would not function. This means a MiniPrep of all the reporter samples, GFP, YFP and CFP; the plasmids 1-3A and 1-5A; and the PUC, PCCI and 5-4E samples. After this, the results in the agarose gel show a very faint result with the DNA of the samples.
Today also we ligated the cuts:
Ligation1:
Backbone 1-5A
Insert1 PUC-57
Insert2 5-4E
Buffer 4uL
Ligase 1uL
H2O mQ 0
Ligation2:
Backbone 1-3A
Insert1 5-14G
Insert2 PCC1
Buffer 4uL
Ligase 1uL
H2O mQ 0
June 13th, 2013
We didn't obtain colonies and we think it is because of the low efficiency of the bacteria. We cloned bacteria to test tubes with LB-medium.
June 14th, 2013
We did miniPreparation of plasmidic DNA.
June 17th, 2013
We cut the DNA's but they were degradated. We innoculated in test tubes again.
June 18th, 2013
We did miniPreparation of plasmidic DNA. And we cut the PCC1 part to see if it liberates the fragment. It didn't work.
June 20th, 2013
We trasnformed the ligation we did the last week to see if now they work with new competent cells.
June 21th, 2013
We obtained colonies of the ligations :D
Image 9. Ligation with the PCC1
Image 10. Ligation with the PUC-57
Main Problems and Further experimentation
We had a lot of problems while working in the lab:
- The BioBricks didn't transformed at the beginning. After some trials we got colonies of the biobricks we wanted to use.
- The genes that were synthesized arrived very late. We had to wait for them to have the complete parts. Anyways we had the biobricks ready to use before the genes arrived.
- One of the genes didn't work, the PCC1. We think it was due to a problem with the restriction site for EcoRI. We arrived to this Conclusion because we made some tests cutting with different enzymes: EcoRI-SpeI and EcoRI-PstI. But it didn't released the fragment. We tried to cut with XbaI and SpeI but there was no DNA available. We are going to continue with this research for the Jamboree presentation.
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