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From 2010.igem.org

Wet lab

09/07/2010 - Day One

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.

12/07/2010 - Day Two

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, the X-blues being significantly more competent than the MG1655s. Repeated the transformation using X-blues 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)

X-blue positive control	100μL cells + 2μL known plasmid solution
X-blue negative control	100μL cells (untransformed)
X-blue + 2009 biobrick	100μL cells + 5μL 2009 biobrick
X-blue + 2010 biobrick	100μL cells + 5μL 2010 biobrick
Nova blue + 2009 biobrick	100μL cells + 5μL 2009 biobrick
Nova blue + 2010 biobrick	100μL cells + 5μL 2010 biobrick

13/07/2010 - Day Three

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 A₆₀₀ value of ~0.5, roughly translating as 2.5x10⁹ 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:

S1	~107 cells/g soil	100μL cell culture plus 900μL LB broth added to soil
S2	~106 cells/g soil	100μL 1/10 dilution of cell culture plus 900μL LB broth added to soil
S3	~105 cells/g soil	100μL 1/100 dilution of cell culture plus 900μL LB broth added to soil
N1	~107 cells/g soil	100μL cell culture plus 900μL LB broth added to soil
N2	~106 cells/g soil	100μL 1/10 dilution of cell culture plus 900μL LB broth added to soil
N3	~105 cells/g soil	100μL 1/100 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.