No growth with the Biobrick B-M/M-B - Transformed bacteria, probable issue with the agar as it looked dry/thin. We still had some bacteria and DNA for a transformation so re-made the plates and decided to try again with the transformations and plating in order to see if they grow on new plates.
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No growth was observed of the cells transformed with the ligation product of B-M/M-B (PCR) in pSB1C3. We suspected an issue with the agar as it looked dry and thin. We still had some bacteria and DNA left from the previous transformation and were able to re-transform with new, fresh agar plates to double check of the transformation was successful.
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. Russell and Rachel plated their transformed bacteria and left to grow over night.
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Russell and Rachel plated their transformed bacteria and incubated them over night.
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. The plates from the previous day's pilot study was viewed. It was found that initially, there was an insufficient number of cells in the culture to plate and hence within the first 3 hours, there were little to no cells on the plates. With progression of time there were more colonies.
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The plates from the previous day's pilot study of PyeaR + GFP was viewed. It was found that initially, there was an insufficient number of cells in the culture to plate and hence within the first 3 hours, there were little to no cells on the plates. With progression of time there were more colonies.
Again, this week was very much centered around lab work and in producing our first BioBricks. We successfully ligated the B-M and M-B inserts into the plasmid backbone. We also began our Growth Study to characterise PyeaR-GFP (BBa_K381001). This has been very much a hectic week but has been totally worth while, seeing the results.
Day 1 (06/08/12)
Research
With the constructs for the comparator circuit designed, research went ahead on how and who we would get the constructs synthesised. Khadija and Pascoe looked at various companies to find one which would synthesise our DNA at the best price but also within a small time frame.
Labs
Russell and Rachel ran their isolated plasmids containing RFP, eCFP and AraC promoter BioBricks on an agarose gel and found that the bands given were not in-keeping with the size they expected them to be. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not, resulting in the abnormal results and it was concluded that further experiments were needed.
A gel of the digested PCR products was rerun following the previous Friday's results. Again the gel was unclear so we decided to repeat the PCR restriction digest.
The PyeaR + GFP BioBrick in pSB1C3, that had been miniprepped last week, was nanodropped. Following Friday, when the restriction digest of the plasmid with EcoRI and PstI was unsuccessful, we repeated the digest to remove the insert from the backbone. Joy used the nanodropped Pyear + GFP sample. Unlike Friday, the digest was successful.
Day 2 (07/08/12)
Research
Continued research into companies for synthesising our DNA. Khadija emailed GenScript for a quote of our order and time it would take for the genes to arrive.
Labs
Russell calculated the expected lengths of the plasmids containing RFP, eCFP and AraC promoter BioBricks in pCB1C3 and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC), however the results in the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1), a stark difference from what was expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert (WHAT ENzYMES), in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the smaples and found that only a very small amount of DNA was present in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:
Clearly a stark difference between the expected result and the actual result. Following this experiment Russell then carried out a DNA mini-prep on the second samples of media containing transformed E. coli jeft in the fridge as a backup; this time using the Bioline Plasmid Isolation kit (Protocol can be found Here). The plasmids that had been isolated from the second DNA mini-prep were then nano-dropped and showed slightly better results, which were:
Despite not being in the 100s as desired we decided that we could use this DNA to carry out future restriction digests and gel electrophoresis in order to validate. We then isolate DNA from the E. coli that was to be transformed by Rachel for the future restriction digest/ligation to produce new biobricks.
Rachel transformed new "E. coli" with the original biobricks in order to produce a new stock of DNA as we had left gaps between using the bacteria and DNA previously which could account for the very low levels of DNA we had experienced in the nanodrops.
Day 3 (08/08/12)
Labs
Rachel checked the transformed bacteria and found that some samples had grown but others hadn't quite as much; they were left to incubate for a few more hours to grow before inoculation.
Russell carried out a restriction digest of each isolated plasmid of which we hoped that they hopefully contained the RFP, eCFP or AraC BioBricks. He did one single digest of each plasmid with just EcoR1 to linerarise the plasmid, and one double digest of each plasmid with EcoR1 + Pst1 to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA that are in the biobricks due to the unusual results previously; if they show fragments of expected sizes then the biobricks transformed into E. coli by Rachel will be used in the ligation of new biobricks and to carry out quantitative experiments.
A plan was made to characterise PyeaR + GFP (BBa_K1001) and thought through and discussed between Lukas, Rebecca and Joy. We plan to grow up PyeaR + GFP in cultures, reading the absorbance at 660nm of the samples regularly (hourly). The inoculation will be made directly from the plate. The pilot study will last for 6 hours and the full study, if the pilot study goes according to plan, will be 12 hours. To generate a comparison of PyeaR + GFP containing cells against non transformed cells, Bioline competent cells were plated and grown for next day use.
The restriction digests (see Friday, Week 4) of the repeated PCR of B-M/M-B was perfromed. Again, it was run on a gel. This time, it showed that the PCR was successful in amplifying the hybrid promoters!
Following validation of the PCR products, we ligated M-B and B-M promoters into pSB1C3. We initially nanodropped each of the samples and used the quantitate data to set up a ligation at a 3:1 ratio between insert to pSB1C3. Positive and negative controls were added to ensure quality control if something did go wrong so that we could determine what caused the error. As s negative control, pSB1C3 would be transformed into alpha gold select cells from Bioline; as a positive control, we ligated PyeaR-GFP into pSB1C3 and transformed the plasmid into alpha cells.
Day 4 (09/08/12)
Research
We finally have our constructs ready to send of for synthesising!
Labs
. Rachel and Russell looked at the transformed bacteria and found that the plates had been infected with something other than the bacteria we were looking for. We had some of the transformant left over so decided to plate again, however the plates that were made were dry/thin. We decided to leave it and make new plates the next day.
. To test whether the ligation had worked, a restriction digest was run. We digested the samples with BamH1 to linearise. If the ligation was successful, the inserts would be in pSB1C3 and hence be 2321bp. If unlinearised, they should travel further due to supercoiling. When linearised they should match this length. Running the products on the restriction digest on a gel showed that we did indeed succeed in ligating B-M and M-B into the iGEM backbone!!!
. The potential BioBricks were transformed into alpha cells and grown overnight. The protocol used followed that on the Lab Protocol page.
. The pilot study was carried out for the planned 6 hours. From the plate a colony was selected and inoculated into 15ml of LB media in a sterile tube. Into this potassium nitrate was added at 0mm, 5mM and 10mM concentration. E.coli cells were inoculated into the same amount of media without the addition of potassium nitrate. Every hour an absorbance reading was taken and a serial dilution was made. Of the dilutions as we do not know the number of cells to be expected 3 of the dilutions were plated (10^-3, 4 and -5).
Day 5 (10/08/12)
Research
After some discussion with advisors into finance, feasibility of time and work, we decided to only send one pair of our constructs to be synthesised.
Labs
No growth was observed of the cells transformed with the ligation product of B-M/M-B (PCR) in pSB1C3. We suspected an issue with the agar as it looked dry and thin. We still had some bacteria and DNA left from the previous transformation and were able to re-transform with new, fresh agar plates to double check of the transformation was successful.
Russell and Rachel plated their transformed bacteria and incubated them over night.
The plates from the previous day's pilot study of PyeaR + GFP was viewed. It was found that initially, there was an insufficient number of cells in the culture to plate and hence within the first 3 hours, there were little to no cells on the plates. With progression of time there were more colonies.
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