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Team:NRP-UEA-Norwich/Week7
From 2012.igem.org
Welcome to the NRP UEA iGEM 2012 Wiki Lab Book
Please choose the relevant link to access our diary of that week!
Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments
Contents |
. Research into sequencing companies to have our BioBricks sequenced.
. Finalising ordering of genes to be synthesised.
. Transformation of CFP, RFP and GFP by Rebecca.
. Plan of requirements for sequencing of B-M and M-B
. Message from GenScript confirming synthesising of ordered genes.
' Plates were found to not be contaminated but there was no growth on any of the plates.
. Retransformed CFP, RFP and GFP into alpha competent cells and plated. Positive controls were made. Some of the transformation culture was retained and more LB medium was added and grown overnight.
. Planning of growth study. Growth Study protocol is on the Lab Protocol page
. The transformation was successful and therefore, inoculations of CFP, RFP and GFP were made.
. Redid the growth study using Alpha cells, PyeaR transformed cells, B-M transformed cells and M-B transformed cells. The experiment lasted 12 hours to give a representative view.
. A restriction digest was carried out by Khadija to both validate and linearise B-M and M-B within the iGEM pSB1C3 backbone. It was linearised in preparation of ligating a reporter to the insert (B-M and M-B hybrid promoter). The restriction digest was carried out using Pst1 and Spe1. This was left overnight to fully complete the digestion.
. Minipreps of M-B and B-M.
. The overnight restriction digest was run on an agarose gel (1% w/v) for an hour and a half. The image showed that MB1, MB2, MB4 and BM2 were fully cut. These showed only one band. The small fragment which would be only a few base pairs long was not seen as expected. Some of the samples ran further, we expect that these are uncut plasmids.
. BioBrick (BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP)) transformants that had been grown in culture were miniprepped (plasmid isolation protocol in lab protocol) by Lukas to attain pure plasmid DNA. This was stored in the freezer.
. As the Growth study carried out the previous day did not show the lag phase as the starting concentrations were too high, a lag phase only study was carried out. This involved starting the cultures at a lower absorbance and hence concentration. The absorbances started at were around the 0.04 marker however, dilution of these to reach an exact 0.04 was very difficult as there were many errors in the machine, therefore, 10µl of cultures grown overnight were pipetted into 1ml of LB broth media in a cuvette. The study lasted for 5 hours with readings taken every hour.
. The primers ordered arrived! A PCR primer stock was made up to 100µM, then further diluted.
. Preparation of plasmid DNA of our BioBricks to be sent for sequencing. 6µL of the DNA was placed into separate eppendorfs to be sent off.
. Rachel carried out a gel purification (on both MB and BM from the gel run) using promega wizzard kit (Ref: A9281) as seen in the lab protocol page. From the gel, the lanes containing BM1, BM2, BM3, BM4, MB1, MB2 and MB4. These refer to bacterial mammalian and the mammalian bacterial promoter transformed cells from different colonies. The purified samples were stored in the freezer.
. Rebecca transformed BioBrick BBa_K561002 (PFDHF +RFP + TETR) into Bioline Alpha Select Gold Standard cells following the transformation protocol as seen in the lab protocols page. The negative control was simply alpha cells, untransformed. The transformation was plated onto agar plates containing chloramphenicol resistance at 25µg/ml.
. Khadija nanodropped the miniprepped plasmid BioBricks BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP). As many cultures were grown per a single colony on the plates, there were separate epppendorfs containing the plasmid DNA. In total there were 4 of CFP, RFP and GFP samples. These were shown to have high concentrations of DNA ranging from 165ng/µL to 332 ng/µL. Following the nanodrop a restriction digest was carried out to validate the plasmid DNA. Two sets of restriction digests were carried out. One is to cut at Pst1 of the flanking suffix/prefix DNA and the other digest is to cut a specific restriction site in the backbone. As BBa_K206009 is found in pSB1AK3 there is a 'Hind'III site and BBa_K081014 is in pSB1C3 and contains an EcoRV site. BBa_E0420 contains many restriction sites but none that can be cut by enzymes that we have in immediate stock. Therefore, a second restriction digest was not carried out on this BioBrick. The single digests involved 1µL DNA, 2µL buffer, 0.2µL BSA, 0.5µL of restriction enzyme and 16.3 µL of distilled water. Buffer H was used in the digests using EcoR1, Buffer B for HindIII and Buffer D for EcoRV. These were stored in the freezer after 2 and a half hours of digestion.
