"
Team:NRP-UEA-Norwich/Week8
From 2012.igem.org
Revision as of 17:24, 24 September 2012 by LukasHarnisch (Talk | contribs)
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 |
This has been the week of restriction digests. We have been working hard on trying to get fluorescent proteins ready for ligating into our BioBricks. Thee BioBricks have also been prepared for sending off to be sequenced. We're super hopeful that the sequenced confirm that they are our BioBricks. We also have the stats for our growth study carried out a couple of weeks ago. Check that out on our experiments page.
Khadija carried out a double digest on CFP, GFP and RFP. They were digested with Pst1 and Xba1 and left overnight. The mastermix was made using 10µl Buffer H, 1µl BSA, 2.5µl Xba1 and 2.5µl Pst1. This was evenly distributed into 5 tubes. The 5 tubes contained: 3µl GFP and 13.8µl of water (nuclease free), 3.94 µl GFP and 12.86 µl water, 3.47 µl RFP and 13.33 µl water, 3.59 µl RFP and 13.21 µl water and 3.6 µl CFP and 13.2 µl of water.
The single digests from the previous Friday were run on a 1% agarose gel. The gel showed that GFP was cut once and linearised, as expected, if cut by either Pst1 or EcoRV. The fragment length was estimated to be around 3000 bp. If the linearised fragment was GFP, it would be around 2800 bp. The digest of the RFP BioBrick produced two fragments. This should not have occurred. We decided to repeat the single digest of RFP.
The single digest of RFP was carried out alongside the single digest of CFP. We decided only to do single digests with Pst1 to validate. The quantities remained the same. 2µl Buffer H, 0.2µl BSA, 0.5µl, 1 µl DNA and 16.3 µl of water. These were left overnight to digest (both the single and double digests) (Figure 1.).
Looking at the plates from Friday which had been stored in the fridge after a day in an incubator, we saw growth on the plates. Both the negative and positive controls showed the expected level of growth. We inoculated the plates containing colonies transformed with the BBa_K561002 (PFDHF +RFP + TETR) into media culture with chloramphenicol. These we left in a shaking incubator overnight at 37degrees. In total three colonies were inoculated into three 5 ml media tubes.
Liaisons with BioSciences continued to buy sequencing vouchers.
A 1% agarose gel was made by Rebecca and poured whilst Khadija began setting up the completed restriction digest products to be run on a gel. 15µl of the single digests were pipetted into 5µl of loading dye whilst 20µl of double digested DNA was mixed with 3µl of loading dye. Into the wells all the prepared samples were loaded. The electrophoresis took place for 1hour and 30mins.
After running the gel, we noticed the bands were not straight. We then noticed the gel itself had not set straight therefore, with inconsistent thickness of the gel, we suspect this could have affected the outcome. We found that whilst the cut out fragments were what we expected them to be ~800bp, the plasmid backbone was evenly 1000bp out. We decided to repeat the digests.
The single digest was completely redone. As originally multiple colonies were inoculated, we decided to digest all the samples we had. In total we digested 11 samples (3 GFP, 5 RFP and 4 CFP). Again the quantities in each were as follows 2µl Buffer H, 0.2µl BSA, 0.5µl, 1 µl DNA and 16.3 µl of water. These were all run overnight.
The grown overnight cultures of PFDHF + RFP + TETR were miniprepped. The concentrations of these have yet to be determined.
A 1% agarose gel was made and the overnight single digests of CFP, RFP and GFP were run on the gel. 2 samples of each were run alongside the uncut versions. The gel confirmed GFP being the BioBrick we were after. The others again showed different lengths, therefore, we can confirm they were not what we predict them to be.
Following this the GFP samples from which the digests were made from underwent a double digest using Xba1 and Pst1. The double digest is separate the GFP insert from the plasmid backbone so that we can isolate the DNA from gel and ligate it to our BioBrick: M-B and B-M. The GFP samples that we ran the double digest on had the following concentrations: GFP1 = 332.9ng/µl, GFP2 = 253.7ng/µl, GFP3 = 255.8ng/µl and GFP4 = 267.1 ng/µl. Using these figures, 3µl of GFP1, 3.94µl of GFP2, 3.9µl of GFP3 and 3.79µl of GFP4 was pipetted into eppendorfs with 3.2µl of a master mix solution consisting of 2.5µl of Buffer H, 1µl BSA, 2.5µl Xba1 and 2.5µl Pst1. Water was added to each of these to make them up to 20µl. This was left overnight.
Following yesterday's minipreps of PFDHF + RFP + TETR, we nanodropped the isolated plasmids. The concentrations were very high. With 3 inoculations made from each colony, these repeats of each colony were labelled a, b and c. The concentrations were as follows:
1a) 656.3ng/µl 1b) 550.1ng/µl 1c) 502.2ng/µl
2a) 423.1 ng/µl 2b) 399.8ng/µl 2c) 347.7ng/µl
3a) 585.0ng/µl 3b) 440.1ng/µl 3c) 522.4ng/µl
As we are low on the pSB1C3 + M-B/B-M to send for sequencing and to iGEM, we ran single and double digests of these. The single digests are to validate existing B-M/M-B and the double digest is to take out the small fragment between Spe1 and Pst1 of the suffix to ligate the reporter GFP into. The single digest involved: BM1, BM2, BM3, MB1, MB2, MB3 and MB4. In each of these digests: 2µl DNA, 0.5µl Pst1, 0.2 BSA, 2µl Buffer H and water to make each sample up to 20µl. The double digest involved 1ng of DNA (this was based on the concentrations of the minipreps obtained from nanodropping. To these 0.5 Xba1 , 0.5µl Spe1, 0.2 BSA, 2µl Buffer H and water to make each sample up to 20µl.
Lukas and Joy ran the double digests and the single digests from yesterday. The double digests of GFP showed that the DNA isolated was not GFP. Despite there being two bands, the fragments were too big. The single and double digests of B-M and M-B showed positive results. There were single clear bands at the expected size distance. These were excised from the gel and purified (Firgure 2.)
Following the success of the double digest of B-M and M-B, the large fragment was cut from the gel and the DNA purified.
The new miniprep of BM and MB was packaged and made ready to be sent to iGEM. The same DNA was sent for sequencing.
As GFP, RFP and CFP that we previously had worked with were not what we expected them to be, we decided to start afresh with new reporter BioBricks. On our plates we found BBa_E0040 and BBa_E1010 which are also GFP and RFP respectively, with RBS sites. We transformed these into Bioline alpha competent cells. Two controls were used: negative control of just competent cells and also a positive control of alpha competent cells transformed with PR8209 which also contains kanamycin that the two BioBricks contain. We did two separate transformations, this was to save time, in case either groups did this wrong. Rachel and Pascoe did one set of transformations and Rebecca and Khadija did another.
The sequencing for our BioBricks M-B and B-M came back. We will need to do some analysis to confirm the sequences. BM3 was found to be the sequence we expected. We will send this to iGEM HQ and hopefully, we have officially made a BioBrick. Most of the sequences confirmed that they were what we expected them to be with the exceptions of the newly made ones. Unfortunately, we do not have sufficient amounts of the other samples to send to iGEM. We are now looking to ask for the sent off samples to be returned.
Statistical analysis into the Growth Study carried out last week shows that the growth rates between Alpha cells (Control) and PyeaR, MB and BM are not significantly different. However, the independent T Test significance level for PyeaR is 0.050 and hence despite not being significantly different at the 95% confidence level, it is significantly different at the 90% confidence. There was also no significant difference between BM and MB. There was however, a significant difference between PyeaR transformed cells and BM and MB.
