Team:NRP-UEA-Norwich/Week4

From 2012.igem.org

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. 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).
. 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).
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The media inoculated media was placed in the 37 °C agitator and left overnight to grow.
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The media inoculated media was placed in the 37 °C agitator and left overnight to grow. The protocol for this can be found [[http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Inoculations Here]]
==Day 3==
==Day 3==

Revision as of 10:37, 8 August 2012

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NRP UEA iGEM 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

Contents

Week 4

Day 1

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 occuring 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 came to visit in order to look at the studio for our film being recorded on Wednesday.

Labs

. Joy and Lukas looked at doing the ligation for our first biobrick using pYeaR/CarG promoter.

. Rebecca transformed the ligated biobricks.


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

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 chlorophenicol 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

The JK-R5-PL Construct

. Again the transformation was unsuccessful.

. Rebecca purified hybrid promoter 2 from gel slice.

. Lukas ran hybrid promoter 2 on gel.

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.

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

. RECEIVED A LETTER FROM DAVID ATTENBOROUGH!

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

. 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.

. Joy, Rebecca and Lukas ran a PCR of the plasmid backbone to get some more.

Day 5

. Ran a gel for PCR and isolated RFP/eCFP/AraC

. Nano-dropped the plasmid DNA

. Restriction digest of insert + primers and plasmid backbone

. Gel electrophesis to confirm plasmid identity