From 2012.igem.org

Revision as of 22:57, 17 September 2012

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

Week 4

This week we made great progress into designing our own constructs for the comparative circuit. The amount of lab work has picked up. We are extremely close to creating our own BioBricks. Hopefully by next week, we will have them.

Day 1 (30/07/12)

Research and Meetings

Khadija and Pascoe found problem over the weekend with stop codon; two options are scaffold or assembly standard 23. Leaning towards the other assembly standard as it's less risky than the scaffold, however discussion occurring on both. They developed two main constructs and each took one; one saw overlapping stands, the other saw strands together. Khadija produced a logic table of codons that could bind to one another. Amy also came to visit in order to look at the studio we bfor our film being recorded on Wednesday.

Labs

. Joy and Lukas began planning for a ligation for our first biobrick using pYeaR/CarG promoter. Following planning, they ligated B-M into the iGEM backbone (pSB1C3).

. Following the ligation, Rebecca transformed the potential Bio-Brick into Bioline alpha select competent cells.

After coming into the lab following growth overnight on the previous Friday to Saturday Russell and Rachel found that the transformed E. coli had not grown as much as they would have liked, therefore the plates were returned to the 37 °C incubator to grow some more overnight.

Day 2 (31/07/12)

Research

. Russell and Rachel looked at nitrite reductase sequences.

. The whole team worked with Khadija and Pascoe on the DNA sequence, four working on Khadija's logic map and three working with Pascoe's idea.

Labs

. As the plates from yesterday's transformations showed no growth, Rebecca redid two of the transformations, this time with both negative and positive controls. She also made more chloramphenicol plates and inoculated more hybrid promoter transformed E.coli into media culture. The transformations from yesterday were incubated for longer in hope that colonial growth would appear.

. Lukas and Joy isolated plasmid DNA from "E.coli" containing the hybrid promoter 1. They then double digested the plasmid using EcoR1 and Pst1. Sac1 and Spe1 was also used as a test for the correct plasmid isolated. This was also carried out on hybrid promoter 2 plasmids. Following this, they ran the digested plasmid fragments of promoter 1 on gel.

After leaving the E. coli that had been transformed with RFP, eCFP and AraC promoter to grow some more overnight there was some success as more colonies were produced on almost all of plates. Russell and Rachel inoculated 5 ml tubes of LB media (+ 5µl ampicillin) with transformed E. coli as follows:

. Two tubes of RFP-transformed E. coli from different colonies on the 200µl plate

. Two tubes of eCFP-transformed E. coli from different colonies on the 200µl plate

. Four tubes of AraC Promoter-transformed E. coli from different colonies on the 200µl plate (due to the fact we need promoters for each of the fluorescent proteins thus we decided to create double the amount).

The media inoculated media was placed in the 37 °C agitator and left overnight to grow. The protocol for this can be found Here

Day 3 (01/08/12)

The JK-R5-PL Construct

Labs

. Rachel and Russell checked the inoculated media and found that it had grown a little bit but not enough for a likely successful experiment, therefore they chose to leave the bacteria to grow some more before attempting a DNA mini-prep.

. Again the transformation with the B-MM and M-B in pUC57 were unsuccessful. The negative control showed no growth and the positive controlled showed growth. The ligation was unsuccessful.

. Rebecca purified B-M insert DNA from the gel slice frozen from the previous night. This was done by using the Promega kit. The protocol is on the lab protocol page.

. Lukas reran B-M on a 1% agarose gel for an hour.

Research

In the morning Khadija, Pascoe, Rebecca and Russell looked at one of the proposed constructs and worked at producing a DNA sequence that would give us two complimentary strands that would bind together once transcribed to mRNA. After a few hours of work and lots of adjustments and edits we finally had our construct which we named the JK-R5-PL construct (using the initials of the team members). The construct is the coming-together of two separate constructs which would be in between the promoter and reporter sequences of the gene we were producing. We were pleased with the JK-R5-PL construct because it meant the ribosome binding site of both original constructs was covered, meaning that the two reporter proteins would not be expressed resulting in the first step of the comparator circuit being completed!

Video

Amy Congdon visited along with two of her colleagues to film for the final presentation video. The artist team had produced some fantastic visual adaptations of cancer cells and the bacteria climbing over them to analyse the environment and act upon the information received. We also filmed the more concept-based aspect of the video, which looks at a future world where comparator circuit-containing bacteria are commonplace in all human beings assessing the body for disease and reporting the results back to the person.

Day 4 (02/08/12)

. RECEIVED A LETTER FROM DAVID ATTENBOROUGH!

. Posters for the Forum returned from the print, ready to start putting them up.

. Joy and Lukas ran PCR on M-B, B-M and a negative control.

. Russell and Rachel worked on the DNA purification and isolated the plasmids for RFP, eCFP and AraC promoter and put on ice to run on an agarose gel the next day.

Russell and Rachel used the media inoculated with E. coli that had been transformed with biobricks of RFP, eCFP or AraC Promoter to isolate the biobricks plasmids using the Promega plasmid isolation kit. They used one tube of media for each biobrick, and left a second tube of media for each biobrick in the fridge to be used as a back-up in case of any problems with the original tubes. Once the plasmids were isolated they were placed in the freezer and left to be run on an agarose gel in order to validate the DNA that had been extracted at a later date.

Day 5 (03/08/12)

. To check the DNA concentrations of B-M and M-B we have purified, nanodropping was used. We found that the DNA concentrations within all the eppendorfs were above 300ng/µl.

. To remove the PCR products such as primers, salt and buffer from the DNA that had undergone PCR, Promega, PCR cleanup kit was used.

. Lukas carried out a double and single restriction digest on PyeaR + GFP (BBa_K381001). The single digest is to be used as a means of validating whether the isolated plasmid DNA is in fact BBa_k381001. This is important as the plasmid DNA can be used as a positive control in ligations and transformations. In the single digest 3µl of DNA, 2µl buffer D, 0.2µlBSA, 0.5µl of EcoRV and 14.3µlof distilled water was used. The double digest was carried out to produce more plasmid backbone. As we had multiple eppendorfs containing plasmid DNA of Pyear + GFP, different diigests were set up containing different amounts of DNA and water. The constants were: 0.4µlBSA, 1µl EcoRV and 1µl Pst1. The following were different in different tubes: 24.5µl DNA (2µg/µl) to 9.1µl distilled water and 23.1µl DNA (1.5µg/µl) to 10.5µl distilled water.

. A double digest was also carried out on B-M and M-B. The enzymes used were EcoR1 and Pst1. This removed the insert from the pUC57 backbone. Using the nanodrop concentrations, the volume of DNA needed was worked out to be 11.2µl and 10.82 µl for B-M and M-B respectively. The distilled water added was 5.6 µl and 6.4µl, respectively. To these 2µl buffer H, 0.2µl BSA, 0.5µl EcoR1 and 0.5µl of Pst1 was added.

. To further validate B-M and M-B, there was another restrictionn digest carried out. A single digest using BamH1.

. Following the restriction digest, the DNA was nanodropped to check the concentrations. The concentrations fell to 88.6ng/µl and 92.2ng/µl for B-M and M-B respectively.

. Gel electrophoresis was used to confirm the identity of the plasmids and for gel purification. The single digest waas successful and thus confirmed that the contents of the eppendorf was in fact PyeaR + GFP that we were trying to isolate. The double digest was not successful and needed to be repeated. On the same gel, the PCR of B-M and M-B was also run. These were run on a 1.4% agarose gel. If the PCR cloning, purification and digestion were succesful, 251bp fragments should be produced. The gel showed shadowing and was not completely clear. The gel will have to be rerun.

. The BamH1 digested products were run on a gel. It showed 200bp fragments, suggesting that the insert was produced.