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Team:NRP-UEA-Norwich/Week7
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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 |
With half the team away for the week, this was a hard week in terms of lab work. The remaining team members, concentrated mainly on doing lab work and in particularly the completion of the growth studies which produced good results which can be analysed. However, there were also many highlights such as the confirmation of our arduously designed gene constructs being made and the arrival of primers allowing us to send our BioBricks to be sequenced.
Research was carried out into companies and their requirements for sequencing. This is preparation for sequencing of our own BioBricks (B-M and M-B in pSB1C3. Sequencing is required to confirm they are the correct sequences before we can send them to iGEM. Lukas found that the requirements for Bio-LifeSciences was 5-6µL of plasmid DNA at a concentration of 100ng/µL and the primers of quantity of 10µL and concentration of 3.2pmol/µL. The primers were also ordered.
The genes for synthesis of our comparator circuit DNA constructs were finalised with GenScript. Witt three weeks left we are hoping they will arrive end of week 9 or start of week 10 to get them sent off in time as new BioBricks.
As the previous transformations of the BioBricks BBa_E0420 (CFP) and BBa_K081014 (RFP) were either unsuccessful or showed contamination, we decided to retry the transformation with a different team member. The procedure used was that seen on the lab protocol page. In addition, a further BioBrick was transformed: BBa_K206009 (GFP). Again, this will also be used in the characterisation of our BioBricks as these will eventually be ligated to our promoter. The transformants of these were plated on ampicillin plates of a concentration of 100µg/ml. A negative control of E.coli cells were plated also on ampicillin plates.
We also made a plan of requirements for sequencing of B-M and M-B. We have already planned for the requirements but here we set aside the DNAs we needed and pipetted them into separate eppendorfs.
A message from GenScript confirming synthesising of ordered genes!
The transformation was unsuccessful, there was no sign of contamination as there were no growth on any of the plates. A transformation of CFP, RFP and GFP into alpha competent cells was repeated. Some changes were made: positive controls were made (plasmids containing M-B with backbones that contain ampicillin resistance and chloramphenicol resistance). Through some slight confusion, GFP had been plated on ampicillin plates the previous day, as the BioBrick contains only chloramphenicol resistance, it was plated on chloramphenicol plates (25µg/ml). The rest of the protocol was unchanged from the previous day. Some of the transformation culture was retained and more LB medium was added and grown overnight.
A growth study involving PyeaR-GFP (BBa_K381001) BioBrick and alpha cells was planned again but this time involving M-B and B-M BioBricks too. The plan is grow these cells in LB medium without antibiotic resistance in sterile conditions overnight to increase the number of cells. The next day, these will be pelleted down to obtain a large number of cells and these will be diluted down with LB both till an absorbance of 0.2 is reached. From then on, these will be run for 12 hours.
Following the plan of the growth study plan set up the previous day, Khadija, Lukas and Rebecca arrived into the labs bright and early and began the growth study. The full experiment details can be found on the experiments page (including statistical analysis and graphical data).
The transformation concerning the GFP, RFP and CFP were successful. There was growth on all the plates except from the negative control plates. Some plates showed greater numbers of colonies than others. The positive control plates showed lots of colonies. The GFP plates showed lots of growth, too. The RFP and CFP showed less growth. There were less than 10 colonies on these plates. Inoculations of these were made into LB media and incubated overnight at 37 degrees Celsius.
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. From previous experiments which ligated M-B and B-M into pSB1C3, this produced 4 B-M's and 4 B-Ms labeled BM1-4 and M-B1-4. These were digested (Figure 1.).
The running inoculations of M-B and B-M BioBricks were miniprepped following the protocols on the lab protocol page. This produced more plasmid DNA. These need to be nanodropped.
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 (Figure 1.)
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! iGEM sequencing primer (VF and VR) stock was made up to 100µM by resuspension in water. These were diluted to match the requirements of Bioscience of 3.2pml/µL. This is sufficient for 10 reactions. 3.2µL of this was further diluted with 96.8µL distilled water to get 100µL of primer at a concentration of 3.2pml/µL. This is sufficient for 10 reactions. Following the arrival and preparation of primers, the plasmid DNA of our BioBricks to be sent for sequencing were prepared. The made DNA was packaged into separate eppendorfs to be sent off. Each eppendorf contained 6µL of the DNA (MB1-4 and BM(1-4) and was labelled. As there was a delay, these were unable to be sent off immediately and were frozen again until possible to be sent off.
Rachel carried out a gel purification (on both MB and BM from the gel run) using Promega Wizard 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 HindIII 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 completed digestions were stored in the freezer after 2 and a half hours of digestion ('#Figure 2.)
