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Team:NRP-UEA-Norwich/Week9
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Contents |
. We looked at the plates of transformant colonies of RFP and GFP that have kanamycin resistance. Both Rebecca's and Rachel's plates for GFP looked a little dodgy. Rebecca's had not grown at all whereas, Rachel's had but the colonies were rather large. The RFP colonies 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 Pst1 and Xba1 to do a double restriction digest of RFP, GFP and CFP that we previously used. We decided that despite the backbone fragment seemingly being too large, we would try it out. Into each eppendorf a 3.2µl mastermix sample of 5µl Pst1, 5µl Xba1, 2µl of BSA ad 20µl Buffer H. There were 9 eppendorf tubes containing: 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.
. At the same time, as we were using isolated plasmid DNA we decided to transform the original DNA into alpha competent silver standard cells from Bioline.
. The synthesised DNA for Constructs 1 and 2 that Khadija and Pascoe had designed a few weeks ago, for 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.
. The plates from the transformation of potentially our next two BioBricks, Construct 1 and 2 were looked at. The plates showed good growth. The negative control showed no growth as expected. The positive control of M-B that was still in pUC57 transformed cells showed good growth. The construct plates showed very good growth and immediately Russell innocuated into LB media containing ampicillin. This was grown for a few hours and we found these were sufficiently cloudy so Rebecca and Khadija reinoculated 10µl of these colonies (4 of Construct 1 and 4 of Construct 2) into 2 new tubes each. In total we have 32 tubes of Constructs 1 and 2. These were all returned to the incubator for further incubation.
. The restricted reporter proteins: GFP, RFP and GFP were run on a 1.2% gel to isolate the fragment. We found that the RFP samples we had were the only successfully cut fragments. The others lanes containing GFP and CFP showed no signs of DNA. Khadija cut the RFP fragments out of the gel and Rachel used the Promega Wizard Kit and protocol to purify the DNA.
. Having had little success with the BioBricks of GFP, RFP and CFP after the original transformation in week 3. We decided to use the original plates to streak the DNA. We located the plates which by now have hardened somewhat. Russell streaked the colonies onto fresh ampicillin plates. These were incubated overnight at 37 degrees celsius.
. As we are running low on ready made LB agar, we made extra supplies. In total we made 7 glasses of 250ml agar which were autoclaved.
. Alpha cells that we had previously plated was inoculated into 5ml LB media ready for setting up a calibration curve for the growth study. These were grown at 37 degrees.
. The samples we requested from Life Biosciences were delivered, unfortunately not all the samples we wanted were returned. We called up to request the rest of the samples. Further liason will occur. In the meantime, with using 1µl of BM1, Rebecca transformed 30µl of Bioline alpha select gold standard cells and incubated them at 37 degrees celsius over night.
. PyeaR + GFP transformed cells that we had previously grown on a plate was inoculated by Russell into culture and incubated overnight at 37 degrees.
. As a follow up on the growth study that we did in week 7, we made a calibration curve using LB media as a zero and measuring absorbances at 600nm. The calibration curve was set up using the alpha cells we had grown overnight. The culture was spun down alike in the growth study. The cells peletted was resuspended in LB media and made up to different concentrations. The concentrations themselves were not important. The absorbances were recorded and the from cuvettes, Rebecca plated 20µl of the culture with 180µl of LB media and grew these overnight in a 37 degree incubator.
. The grown overnight cultures of Constructs 1 and 2 were miniprepped by Rebecca and Khadija. These were then nanodropped. Thee following concentrations were found: (C1 = Construct 1, C2 = Construct 2. The following number refers to the isolate colony which was marked on the plate and the 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: 900.0 ng/µl
C2-1b: 633.0 ng/µl
C2-2a: 448.9 ng/µl
C2-2b: 546.9 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 and a new record by Rebecca, we set up a restriction digest to isolate the constructs to ligate into pSB1C3. The double restriction used EcoR1 and Pst1. A master mix was set up using: 4µl of BSA, 40µl of Buffer H, 10µl of EcoR1 and 1µl of Pst1. Into eppendorfs labelled with what Construct DNA, 3.2µl of master mix was put in. These eppendorfs had DNA and nuclease free water pippetted in, in advance. The following amounts of DNA were added and water was added to make the samples up to 20µl.
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
. Additional Agar and LB media was made and autoclaved. The LB media was pipetted into 5 ml bottles before autoclaving.
. Looking at the plates that we transformed yesterday, we saw that the plates were good. The 20µl and 200µl plates of BM1 transformed E.coli contained many colonies. These colonies were compared to those on the control plates. The negative control of just cells showed no colony growth. The positive control of PyeaR + GFP transformed cells, which also contain chloramphenicol resistance showed similar growth to that of the BM1's. From these plate 4 colonies were inoculated into LB media containing chloramphenicol resistance.
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