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Week 9

So many minipreps, it's been crazy! We crowned a new queen of miniprepping, but how long this record will last we don't know since we have a lot more lined up. Down to more serious matters, this week the comparator circuits (Comparator Circuit 1 and 2) have been the centre of attention. The DNA arrived Monday and immediately the pressure was on to get them cloned into pSB1C3 and have them sent off. In addition to this we have managed to get the RFP and CFP BioBrick working through identifying the original plates and colonies and streaking these. It's been a very productive and long week, in both working hours and anticipation.

Day 1 (3/09/12)

We observed the transformant colonies of RFP and GFP that had kanamycin resistance. Both Rebecca's and Rachel's colonies of GFP looked unusual in morphology. Rebecca's had not grown at all whereas Rachel's had but the colonies were rather large. The RFP colonies on the other hand looked good. However after double checking in the parts registry, we realised that these BioBricks have no RBS and therefore we decided to keep these plates for now and went back to the previous BioBricks used with RFP, GFP and CFP.

Russell, Rebecca and Khadija used PstI and XbaI to set up a double restriction digest of RFP, GFP and CFP that we previously used. We decided that despite the backbone fragment being seemingly too large, we would try to use it. Into each sample 3.2µl of mastermix was added. There were reaction set up in total, consisting of 3.6µl of DNA and 13.2µl of water (CFP1), 6.03µl of DNA and 10.77µl of water (CFP2), 3.47µl of DNA and 13.32µl of water (RFP1), 3.92µl of DNA and 12.88µl of water (RFP2), 3µl of DNA and 13.8µl of water (GFP1), 3.91µl of DNA and 12.89µl of water (GFP3) and 3.74µl of DNA and 13.06µl of water (GFP4). These were left overnight to complete reaction.

At the same time, as we were experimenting with the isolated plasmid DNA, we decided to transform the original DNA from the iGEM 96 well plates into alpha competent silver standard cells from Bioline. This was intended to ensure a back up in case the DNA being digested at the moment proved faulty.

The synthesised DNA of Constructs 1 and 2 of the comparator circuit finally arrived!. Immediately, we got to work on it. Rebecca rehydrated the DNA and transformed it into Bioline alpha select gold standard competent cells. Plates were made and incubated overnight at 37 degrees.

Day 2 (4/09/12)

The plates from the transformation of our potential next two BioBricks, Construct 1 and 2, were observed. The colonies showed good growth. The negative control showed no growth as expected. The positive control of MB, that was still in pUC57, showed good growth. The construct plates showed very good growth and Russell innocuated into LB media containing ampicillin. The sample was grown for a few hours and were found to show sufficient cloudy bacterial growth for Rebecca and Khadija to reinoculated 10µl of these the samples (4 of Construct 1 and 4 of Construct 2) into 2 new tubes each. In total we were left with 32 tubes of Constructs 1 and 2. These were all returned to the incubator to be left overnight.

The restricted reporter proteins GFP, RFP and GFP were run on a 1.2% agarose gel to separate the fragments. We found that the RFP samples we had been using were the only successfully cut fragments. The others lanes containing GFP and CFP showed no signs of DNA. Khadija purified the RFP DNA fragments and the sampels were stored in the fridge for ligation later on.

Having had little success with the BioBricks GFP, RFP and CFP following their initial transformation in week 3, we decided to use the original plates to streak the DNA. We located the plates which showed by now hardend media. Russell transferred some colonies onto fresh ampicillin plates incubated them overnight at 37 degrees celsius.

As we are running low on ready made LB agar, we made up extra supplies. In total 7 glasses of 250ml agar were prepared to be autoclaved.

Alpha cells, that we had previously plated, were inoculated into LB broth media to have them ready for usage in creating a calibration curve for growth studies. These were grown at 37 degrees.

Day 3 (5/09/12)

From the streaked RFP and CFP plates, Russel inoculated 6 colonies of RFP and 6 colonies of CFP into LB media of adequate ampiciilin concentration. The samples were grown overnight at 37 degrees.

Transformation grown overnight of BM1

The sequence sampels we had requested from Life Biosciences, were delivered. Unfortunately not all initial tubes that were send off were returned to us. We got in contact with Life Bioscience to inquire about the whereabouts of the missing ones. Further liason was promised. In the meantime Rebecca transformed 30µl of Bioline alpha select gold standard cells with 1µl of BM and incubated the cells at 37 degrees celsius over night.

PyeaR + GFP transformed cells, that had previously been grown on plates, were inoculated by Russell into LB broth and incubated.

As a follow up on the growth study we conducted in week 7, we prepared a calibration curve using LB media as a zero reading and measuring absorbances at 600nm. The calibration curve was set up using the alpha cells we had grown overnight. The culture was spun down in reference to the protocol used growth. The cell pellet was resuspended in LB media and made up to different concentrations. Absorbance readings were recorded and from each sample cells were plated 20µl of the culture and left to grow overnight in a 37 degree incubator.

We identified the plate containing the MB (in pSB1C3) transformed colonies which we originally inoculated and attained the MB1-4 samples that we had sent off for sequencing. We inoculated the colony which later became MB2 and was proven by sequencing to be what we expected. Along with the mentioned colony, 9 others were inoculated. As MB1, 3 and 4 cell did not have the desired DNA sequence, they were discarded. Only the corresponding colonie to sample MB2 was inoculated. All other samples were from new colonies. Later these tubes were reinoculated into a second set of tubes so that in the end we had 20 promising samples. These were all incubated at 37 degrees overnight.

The overnight cultures of Constructs 1 and 2 were miniprepped by Rebecca and Khadija. The isolated DNA were nanodropped. The following concentrations were recorded: (C1 = Construct 1, C2 = Construct 2. Number refer to the isolate colony which was marked on the plate, a and b refer to the tubes we used during mini prepping (2 5ml inoculations were miniprepped into one eppendorf- essentially a and b should contain the same DNA. We separated them due to the protocol stating using 1-10ml)

C1-1a: 523.9 ng/µl

C1-1b: 421. 2ng/µl

C1-2a: 456.5 ng/µl

C1-2b: 422.8 ng/µl

C1-3a: 571.8 ng/µl

C1-3b: 389.0 ng/µl

C1-4a: 438.0 ng/µl

C1-4b: 316.6 ng/µl

C2-1a: 448.9 ng/µl

C2-1b: 546.9 ng/µl

C2-2a: 900.0 ng/µl

C2-2b: 633.0 ng/µl

C2-3a: 572.5 ng/µl

C2-3b: 530.7 ng/µl

C2-4a: 617.7 ng/µl

C2-4b: 548.7 ng/µl

Spurred on by the high concentrations of DNA and a new record held by Rebecca, we set up a restriction digest to isolate the constructs to prepare them to be ligated into pSB1C3. The double restriction used EcoRI and PstI. A master mix was set up using 4µl of BSA, 40µl of Buffer H, 10µl of EcoRI and 1µl of PstI. To each DNA sample, 3.5µl of master mix was added and filled with nuclease free water to each contain 20µl of solution. The following amounts of DNA were added to each sample respectively:

C1-1a: 1.91 µl

C1-1b: 2.37 µl

C1-2a: 2.19 µl

C1-2b: 2.37 µl

C1-3a: 1.75 µl

C1-3b: 2.57 µl

C1-4a: 2.28 µl

C1-4b: 3.16 µl

C2-1a: 2.23 µl

C2-1b: 1.83 µl

C2-2a: 1.11 µl

C2-2b: 1.58 µl

C2-3a: 1.75 µl

C2-3b: 1.88 µl

C2-4a: 1.62 µl

C2-4b: 1.82 µl

The restriction digests was left in a 37 degree water bath overnight.

Additional Agar and LB media was made and autoclaved. The LB media was pipetted into 5 ml bottles before autoclaving.

Day 4 (6/09/12)

Observing the plates that were transformed yesterday, we concluded that the experiment was successful. The 20µl and 200µl plates of BM1 transformed E. coli contained a great amount of colonies. The presented colonies were compared to those on the control plates. The negative control of non transformed alpha cells showed no colonial growth. The positive control of PyeaR + GFP transformed cells, having chloramphenicol resistance, showed similar growth to the one of the BM1 colonies. From these plates, 4 colonies were inoculated into LB media containing chloramphenicol.

The overnight inoculations of MB were miniprepped. A sample of 10µl of the originally inoculated cultures were reinoculated into new media to maintain an ongoing culture. As we were rushing to have MB2 sent for sequencing, we miniprepped MB2 and had in nanodropped first. The concentration was found to be 58.2ng/µl.

The restricted DNA of Constructs 1 and 2 were run on an agarose gel to isolate the desired fragment. Due to insufficient amounts of agarose, only one gel was prepared and as we had 16 samples and a ladder to run, only the samples of prime quality were run (e.g C1-1a, C2,4a, etc). The remainders were stored in the fridge. The gel was prepared to be of 1.5% w/v as the fragment to be isolated was only 171bp. The electrophoresis was run for an hour and it was found that the the ladder had not separated out. For each sample of DNA, 5µl of loading dye was added to 20µl of DNA. As we had gotten hold of a new ladder (100bp ladder), we were unaware that loading dye was not already added. Therefore, there the ladder fragments were not visible on the gel. We were also perplexed by the presence of 3 bands of each sample, though the lowest we suspect to be shadowing. Also two samples of Construct 1 did not successfully digest (C1-2a and C1-3a). The rest showed the expected results. We cut the DNA fragments from the gel and stored them in the fridge overnight to ge purified the next day.

Russell reinoculated a vast amount of RFP and CFP. The original tubes were split and another series of samples were inoculated. This resulted in a totaling of 3 tubes for each colony (total of 6 colonies in tubes for each of RFP and CFP)

Day 5 (7/09/12)

Dry Lab

Joy researched the amount of DNA we would need to send off for sequencing and calculated the dilutions we needed to use for our samples to prepare them to be send off.


The miniprepped MB DNA was nano dropped and the following concentrations were found:

MB1: 78 ng/µl

MB3: 169.3 ng/µl

MB4: 91.7 ng/µl

MB5: 113.0 ng/µl

MB6: 68.2 ng/µl

MB7: 79.2 ng/µl

MB8: 32.2 ng/µl

MB9: 132.2 ng/µl

MB10: 147.6 ng/µl

MB6 and MB 8 had abnormally high 260/2800 readings at 4.19 and 22.85 respectively.

Restriction digest of Comparator Circuit 1 and 2 with Pst1 and EcoR1 with the Promega 1kb ladder

With more agarose available, we prepared a 1.5% w/v gel and the rest of the Construct 1 and 2 samples were run on a gel for an hour. This time, loading dye was added to the ladder. For each sample of DNA, 5µl of loading dye was mixed with 20µl of DNA/ladder. The ladder ran successfully but we did notice the mentioned shadow of each band. Counting the number of bands and their relative distances we could be sure that the last band was shadowing as the shortest marker fragment was also shadowed. The fragments were the right size and these were cut out before being purified.

The gel slices of Construct 1 and 2 fragments were purified using Promega Clean Up kit. After purification the sampels were nanodropped and the follwoing concentrations were as follows:

C1-1a: 15.1 ng/µl

C1-1b: 4.5 ng/µl

C1-2b: - 9.0 ng/µl

C1-3b: - 7.2 ng/µl

C1-4a: 14.0 ng/µl

C1-4b: - 0.4 ng/µl

C2-1a: 7.6 ng/µl

C2-1b: 1.1 ng/µl

C2-2a: 6.3 ng/µl

C2-2b: 0.4 ng/µl

C2-3a: 14.6 ng/µl

C2-3b: 2.2 ng/µl

C2-4a: 14.0 ng/µl

C2-4b: -3.2 ng/µl

All samples containing less than 4ng/µl of DNA were discarded.

Due to the low DNA concentrations of Construct 1 and 2, Rebecca repeated the double restriction digest. However, as there was a slight communication error, the wrong enzymes were added. Instead of PstI and EcoRI, PstI and XbaI was added instead. After 5 hours of digestion, the samples were refrigerated in case they would be needed for later cloning. After further discussion with advisors, we decided the the concentrations ascertained should be sufficient to carry out ligations.

The PyeaR samples that were inoculate on Wednesday, were plated and incubated at 37 degrees overnight. This is in preparation for next week when we hope to characterise the PyeaR + GFP BioBrick further by ascertaining results of growth in potassium nitrate through the use of a fluorimeter.

Rachel miniprepped the BM1 that had been inoculated by Russell on Thursday. Before miniprepping, 5µl was removed and reinoculated. The next day these reinoculations would be taken out of the incubator and placed in the fridge. The miniprepped DNA using the Promega DNA isolation kit was nanodropped and found that there was very little DNA. The was 13.1ng/µl. Again after discussion with advisors, we feel that this may be sufficient and a ligation will be carried out on Monday.

Khadija miniprepped all the RFP and CFP's. In totally 3 bottles of each colony was spun down into a pellet and then the rest followed the protocol of the Promega kit. Following the miniprep all these samples of isolated DNA was nanodropped to identify the concentration. The following concentrations were found:

RFP1 (1): 113.9 ng/µl

RFP1 (2): 123.9 ng/µl

RFP1 (3): 126.1 ng/µl

RFP2 (1): 67.1 ng/µl

RFP2 (2): 201.8 ng/µl

RFP2 (3): 107.7 ng/µl

CFP1 (1): 239.3 ng/µl

CFP1 (2): 246.4 ng/µl

CFP1 (3): 170.9 ng/µl

CFP2 (1): 48.4 ng/µl

CFP2 (2): 218.0 ng/µl

CFP2 (3): 163.9 ng/µl