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Team:University of Sheffield /Wet Lab
From 2008.igem.org
| Introduction | Our project | Modelling | Wet Lab | Our team | Timetable | Miscellaneous |
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| Introduction | Protocols |
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Wet Lab
Contents |
Lab Books
Protocols
Tecan Fluorescence Measurement
Protocol used for characterization :
1. Grow 5ml of overnight cultures of DH5-alpha cells containing the plasmid with GFP-LVA.
2. Resuspend the overnight cultures in 50 ml of LB medium until the OD600 reaches about 0.6.
3. Quickly add 0.2 mM of IPTG to the medium and plate that into 96 well plate.
4. To 96 well plate add 180 ul of LB + IPTG as a control, 180 ul of DH5-alpha cells not induced with IPTG, finally add 180 ul of DH5-alpha induced with IPTG. Carry out the aforementioned in duplicates.
5. Use the high flow cytometry machine Tecan to measure the fluorescence every 15 minutes for 8 hours (excitation wv – 485 nm, emission wv- 535 nm )(including shaking = aeration of the cultures 2 minutes prior each measurement)
Timetable
Dry Lab Period
February 2008
The very beginning. Three team members, Gosia, Eva and Dmitry met each other to discuss the possibility of joining iGEM competition this year. Straight after that Rosie joined the team. March 2008 – The idea to participate in iGEM was presented in the Department of Molecular Biology and Biotechnology (MBB) in the students and staff committee meeting. The head of MBB Department, Prof. D. Hornby agreed to help us in managing this project.
April 2008
- The team now consisted of 9 members, mostly joined by MBB Level 2 students. The early brainstorming took place. However, no ideas that would be feasible to complete during the summer and would have actual impact on science appeared.
- A new science orientated society was established within the University of Sheffield Union of Students. The initial purpose of the SynBio society was to help students within the University to participate in iGEM next year, along with that a Scientific Journal Club with monthly meetings is on the agenda
- Applications for Funding to various sources were sent out.
June 2008
- the idea of sensing biological water contamination employing the Quorum Sensing mechanism was finalised
- 15th June - meeting with Prof. P. Wright took place to discuss a possibility to join labs in the Bioincubator, a new business and research facility in Sheffield
- 29th June - two level 2 students representing the Department of Automatic Control and Systems Engineering joined the team to cover the Engineering parts of the project.
- 30th June - meeting with Dr. Catherine Biggs from the ChELSI group took place to discuss a possible approach towards using the Quorum sensing as a way of sensing water contamination
July 2008
- 2nd to 16th July - team members representing biological and engineering sides of the project introduced each other to their plans of action as well as explained the material behind it.
- 7th July - meeting with Prof. Marian Gheorghe from Automatic Control and Systems Engineering. The meeting discussed the approach towards mathematical modeling of the Quorum Sensing using E.coli as a tool.
- iCHEME offered a financial contribution towards the trip to Jamboree.
- Discussions with the group as well as Dr. C. Biggs, with an aim to choose the right model organism to represent water pathogens.
- Ph.D. student Esther Karunakaran proposed using a fusion histidine kinase as a receptor for V. cholerae autoinducer . V. cholerae , one of the causative agents of water borne diseases, was then chosen as a model organism representing water pathogens.
- The native E.coli BarA - UvrY signal transduction pathway was selected for "hijacking" by our fusion kinase.
- 20th July - students discussed a possible target for GFP insertion in the E.coli genome, the pgaABCD operon was chosen.
- Prof. P. Wright and Dr. C. Biggs representing the ChELSI research group within the University of Sheffield approved the project plan and agreed to provide lab materials and lab space in the Bioincubator research facility.
- IDT DNA kindly agreed on a contribution of £1000 towards the cost of gene synthesis and primer orders. The CqsS – a native receptor for CAI-1 molecules was ordered for further experiments.
August 2008
- 4th August - meeting with David Wengraf to discuss possible biohazard before final approval to enter Bioincuabotr Labs.
- 4th and 5th August - meetings with Prof. Rice so as to choose a correct place to cut the CqsS and BarA to have a functional fusion kinase.
Wet Lab Period
Mid - August 2008
- 7th August - team entered the labs to start actual experiments. Preparation for gene knockout using Datsenko and Wanner method was the first on the agenda. Prof. Stafford from the Univeristy of Sheffield Medical School provided us with electroporation protocol. From 11th August to mid September the electroporation was carried out unsuccessfully.
- 15th August - an official project presentation to the ChELSI research group.
- the transformation using the Biobrick BBa_I763004 containing a plasmid with GFP – LVA tag was made. All attempts were unsuccessful despite using pre – approved protocols and cultures.
- 26th August - Mr. Peter Grant representing the Biofusion Group provided funding to cover the costs of the U. S. VISA for 3 team members.
September 2008
- The gene order for CqsS membrane receptor arrived.
- Along with that Prof. B. Bassler from Princeton University sent us the plasmid containing CqsA - a protein producing CAI -1 (Cholera Autoinducer – 1) with the appropriate purification protocol.
- Prof. O. Melefor from Karolinska Instutue kindly provided us with a DH5 – alpha strain containing a barA knockout
- Prof. Robert Poole agreed to cover further funding for the trip to USA. On 16th September the hotels and flights to the Jamboree were booked, along with registration confirmation of all attending team members.
- The electroporation and transformation were still unsuccessful even after extensive troubleshooting.
- 29th September VISAs were attained.
October 2008
- Plasmid prep of one possible GFP - LVA transformant colony was made. The attempt was to characterize a possible Biobrick using Tecan as well as loading a restriction digest of the plasmid prep on the agarose gel. The results suggested that the colony was most likely contamination.
- 8th to 15th October -preparation for Biobrick submission to the Parts Registry took place. Failed transformations raise suspicions of a faulty Biobrick booklet. Thorough investigation of various DNA parts from all over the booklet proves this hypothesis. Due to faulty DNA shipment no parts can be submitted to the Registry as the deadline for variants was 8th October.
