Latest revision as of 22:08, 27 October 2010

BCCS-Bristol
iGEM 2010

Wet lab

• Achievements • Part Design • Lab Work • Beads • Safety • Improvements •

Our Labspace

Our main achievement in the wet lab has been the creation of a new composite BioBrick, engineering E. coli to produce GFP in response to Nitrates. Not only is this part complete, but it's also well characterised both on its own and working in tandem with a second BioBrick.

We also developed a way of encapsulating our bacteria, allowing an easy and safe method of spreading them in the environment, and also dramatically improving signal detection and E. coli survival in soil.

Finally we worked to further characterise the PyeaR promoter submitted by Edinburgh in 2009, improving the quality of information available in the parts registry.

In this section you can find out more details on all our achievements. You can also find information about about the design of our parts, including all the decisions we made and why, as well as all the information relating to their construction and characterisation. It also contains material on our new encapsulation methods, and finally lists all our safety considerations both in the lab and on a wider environmental scale.

@@ Line 1: / Line 1: @@
 {{:Team:BCCS-Bristol/Header}}
+=Wet lab=
-== Wet lab ==
-'''09/07/2010 - Day One'''
+<center> • [[:Team:BCCS-Bristol/Wetlab/Achievements|Achievements]] • [[:Team:BCCS-Bristol/Wetlab/Part_Design|Part Design]] • [[:Team:BCCS-Bristol/Wetlab/Experiments|Lab Work]] •
+[[:Team:BCCS-Bristol/Wetlab/Beads|Beads]] • [[:Team:BCCS-Bristol/Wetlab/Safety|Safety]] • [[:Team:BCCS-Bristol/Wetlab/Improvements|Improvements]] • </center>
-Made two different competent strains of ''E.coli'' - henceforth referred to as MG1655s and X-blues, and attempted transformations on both using BBa_I13522 (pTet GFP) from the kit - prepared 6 agar plates: 1 positive control for each strain, 1 negative control for each strain and 1 test transformation plate for each strain. The positive control was carried out using a plasmid of known concentration. Ampicillin was used for selection. Plates left overnight.
+[[Image:Picture_018.jpg|right|thumbnail|250px|Our Labspace]]
-'''12/07/2010 - Day Two'''
+Our main achievement in the wet lab has been the creation of a new composite BioBrick, engineering ''E. coli'' to produce GFP in response to Nitrates. Not only is this part complete, but it's also well characterised both on its own and working in tandem with a second BioBrick.
-No growth on test plates. However, growth was observed on the positive control plates for both strains used, indicating both strains of cell used were competent, one being significantly more competent than the other. Repeated the transformation using the more competent strain plus a commercially obtained strain of very high competence, henceforth referred to as Nova Blues (no control plates were made with these cells). A larger amount of BBa_I13522 from both the 2009 and 2010 distribution kits was used for each transformation. Plates left overnight (see table below for details of transformations)
+We also developed a way of encapsulating our bacteria, allowing an easy and safe method of spreading them in the environment, and also dramatically improving signal detection and ''E. coli'' survival in soil.
-{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000"
-!style="border-style: solid; border-width: 1px"| X-blue positive control
-!style="border-style: solid; border-width: 1px"| 100μL cells + 2μL known plasmid solution
-|-
-!style="border-style: solid; border-width: 1px"| X-blue negative control
-!style="border-style: solid; border-width: 1px"| 100μL cells (untransformed)
-|-
-!style="border-style: solid; border-width: 1px"| X-blue + 2009 biobrick
-!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2009 biobrick
-|-
-!style="border-style: solid; border-width: 1px"| X-blue + 2010 biobrick
-!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2010 biobrick
-|-
-!style="border-style: solid; border-width: 1px"| Nova blue + 2009 biobrick
-!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2009 biobrick
-|-
-!style="border-style: solid; border-width: 1px"| Nova blue + 2010 biobrick
-!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2010 biobrick
-|}
+Finally we worked to further characterise the PyeaR promoter submitted by Edinburgh in 2009, improving the quality of information available in the parts registry.
-'''13/07/2010 - Day Three'''
+In this section you can find out more details on all our achievements. You can also find information about about the design of our parts, including all the decisions we made and why, as well as all the information relating to their construction and characterisation. It also contains material on our new encapsulation methods, and finally lists all our safety considerations both in the lab and on a wider environmental scale.
-Achieved growth of cells with the Nova blues only. Viewed cells transformed with the 2009 biobrick under UV light the colonies fluoresced green, indicating GFP expression. Colonies from both 2009 and 2010 transformations were selected and re-plated to grow overnight.
-'''14/07/2010 - Day Four'''
-Re-plated colonies grew successfully, and appear green under normal light. Selected example colony was placed in 50mL LB broth + Ampicillin and left overnight in preparation for miniprep of the bacteria to extract biobrick.
-'''15/07/2010 - Day Five'''
-Combined 3mL of cell culture in 1.5mL eppendorf tube 4 times - total of 12mL combined cell culture. Performed a miniprep on the combined culture using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 200μL of solution containing BBa_I13522 biobrick in high concentration.
-Performed transformation of MG1655 ''E.coli'' cells with 10μL eluted DNA solution per 100μL cells plus a negative control plate. Left overnight.
-'''16/07/2010 - Day Six'''
-Achieved growth of transformed MG1655 cells - a proverbial lawn of green ''E.coli'' was the result of overzealous use of the miniprep DNA solution. Example colonies replated and left to grow in a 30°C oven over the weekend.
-'''19/07/2010 - Day Seven'''
-Removed plates from 30°C oven and placed in fridge.
-'''20/07/2010 - Day Eight'''
-Colonies from the re-plated MG1655s selected and placed in 5mL LB broth + 5μL Ampicillin in preparation for testing on sample soil. Left overnight.
-'''21/07/2010 - Day Nine'''
-Sample soil taken from local source (team member's garden). 6 x ~15g aliquots of soil were placed in 50mL centrifuge tubes, taking care to avoid including wildlife. 3 of the tubes were then sterilised using an autoclave. Meanwhile, a 1 in 10 dilution of the overnight culture of MG1655 cells + BBa_I13522 was found to have an A<sub>600</sub> value of ~0.5, roughly translating as 2.5x10<sup>9</sup> cells/mL in the original culture. The following table lists the rough figures for the 6 soil experiments set up. The lines notated with "S" were for the sterilised tubes, the lines notated with "N" were for the non-sterilised tubes:
-{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000"
-!style="border-style: solid; border-width: 1px"|S1
-!style="border-style: solid; border-width: 1px"| ~10<sup>7</sup> cells/g soil
-!style="border-style: solid; border-width: 1px"| 100μL cell culture plus 900μL LB broth added to soil
-|-
-!style="border-style: solid; border-width: 1px"|S2
-!style="border-style: solid; border-width: 1px"| ~10<sup>6</sup> cells/g soil
-!style="border-style: solid; border-width: 1px"| 100μL <sup>1</sup>/<sub>10</sub> dilution of cell culture plus 900μL LB broth added to soil
-|-
-!style="border-style: solid; border-width: 1px"|S3
-!style="border-style: solid; border-width: 1px"| ~10<sup>5</sup> cells/g soil
-!style="border-style: solid; border-width: 1px"| 100μL <sup>1</sup>/<sub>100</sub> dilution of cell culture plus 900μL LB broth added to soil
-|-
-!style="border-style: solid; border-width: 1px"|N1
-!style="border-style: solid; border-width: 1px"| ~10<sup>7</sup> cells/g soil
-!style="border-style: solid; border-width: 1px"| 100μL  cell culture plus 900μL LB broth added to soil
-|-
-!style="border-style: solid; border-width: 1px"|N2
-!style="border-style: solid; border-width: 1px"| ~10<sup>6</sup> cells/g soil
-!style="border-style: solid; border-width: 1px"| 100μL <sup>1</sup>/<sub>10</sub> dilution of cell culture plus 900μL LB broth added to soil
-|-
-!style="border-style: solid; border-width: 1px"|N3
-!style="border-style: solid; border-width: 1px"| ~10<sup>5</sup> cells/g soil
-!style="border-style: solid; border-width: 1px"| 100μL <sup>1</sup>/<sub>100</sub> dilution of cell culture plus 900μL LB broth added to soil
-|}
-The soil cultures were left overnight at 37°C.
-'''22/07/2010 - Day Ten'''
-To be finished.
-==Safety Issues==
-As the idea of communication between a certain population (in this case E.coli) could raise issues in health and safety of the general public the following precautions were taken during the implementation of the project in the laboratory:
-*Novel proteins handled (FhuA,OsmE,Fiu) were derived from DNA by gene cloning with PCR from E.coli MG1655 a laboratory strain with no toxic implications.
-*Novel proteins handled where screened from the literature to ensure that they will have no toxicity effects.
-*Experiments were implemented in a Level 1 Laboratory with access only by trained individuals.
-*Students involved in experimental work in the laboratory were trained to an appropriate level  to apply relevant techniques and use relevant equipment  and where suitable were supervised whilst carrying out laboratory work.
-*Laboratory workers were always clothed in appropriate manner (lab coat, gloves, safety spectacles).
-*Laboratory workers sterilised their hands before and after laboratory work and before entering and exiting the lab at all times.
-*No bacterial cultures exited the laboratory unless they were suitably packaged and accompanied by one of the team members whilst in transport and this only occurred where it was necessary to transport cultures from one laboratory to another.
-*No purified DNA or biological material was left unattended at any time, and all DNA and biological material was suitably stored according to Level 1 Laboratory rules.
-*Biosafety guidelines where followed under the BCCS-Bristol iGEM'09 supervising team and such guidelines fall within the description of a project that holds approval by the iGEM supervisor Dr.Nigel Savery.