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From 2010.igem.org

Wet lab

For a complete list of the biobricks used during our project, plus a brief description on their use, click here.

09/07/2010 - Day One

Made two different competent strains of E.coli - MG1655s and XL1-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 XL1-blues being significantly more competent than the MG1655s. Repeated the transformation using XL1-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)

XL1-blue positive control	100μL cells + 2μL known plasmid solution
XL1-blue negative control	100μL cells (untransformed)
XL1-blue + 2009 biobrick	100μL cells + 5μL 2009 biobrick
XL1-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 2010 colony was placed in 50mL LB + 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 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 + 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 added to soil
S2	~106 cells/g soil	100μL 1/10 dilution of cell culture plus 900μL LB added to soil
S3	~105 cells/g soil	100μL 1/100 dilution of cell culture plus 900μL LB added to soil
N1	~107 cells/g soil	100μL cell culture plus 900μL LB added to soil
N2	~106 cells/g soil	100μL 1/10 dilution of cell culture plus 900μL LB added to soil
N3	~105 cells/g soil	100μL 1/100 dilution of cell culture plus 900μL LB added to soil

The soil cultures were left overnight at 37°C.

22/07/2010 - Day Ten

Soil samples viewed under fluorescence microscope. Controls used were as follows:

A 200μL aliquot of pure water

A 200μL aliquot of cell culture expressing GFP

A sample of the unsterilised soil

A sample of the unsterilised soil with a 200μL aliquot of cell culture applied 10 minutes before visualisation

All test soil samples showed growth of E.coli expressing GFP. As might have been expected, a significant increase in growth was observed in the sterile samples when compared to the non-sterile samples at similar dilutions of bacteria/g soil. Click here for some example images from the test.

23/07/2010 - Day Eleven

Concept meeting.

26/07/2010 - Day Twelve

Plated out 2 strains of E.coli, one containing the biobrick BBa_K216005 and the other containing BBa_K216009. Left overnight.

27/07/2010 - Day Thirteen

Selected colonies from the plates of E.coli containing the biobricks BBa_K216005 and BBa_K216009 placed in 50mL LB + 50μL Ampicillin. Left overnight.

Biobricks BBa_E0840, BBa_E0240 and BBa_J04450 were taken from the kit and used to transform Nova Blue E.coli cells. Plated out on agar + Ampicillin for BBa_E0840 and BBa_E0240 transformations, and agar + Kanamycin for BBa_J04450 transformation. Left overnight.

28/07/10 - Day Fourteen

Combined 3mL of BBa_K216005 and BBa_K216009 cell cultures in 1.5mL eppendorf tubes twice - total of 6mL combined cell culture for each biobrick. Performed a miniprep on the combined cultures using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 100μL of solution containing BBa_K216005 and BBa_K216009 biobricks in high concentration.

Performed transformation of XL1-Blue E.coli cells with 2μL eluted BBa_K216009 solution per 100μL cells. Left overnight.

Selected colonies from the Nova blue E.coli plates with the biobricks BBa_E0840, BBa_E0240 and BBa_J04450 were taken and placed in 5mL LB + 5μL Ampicillin. Left overnight. Writing this, realised that the cells containing the BBa_J04450 biobrick are Kanamycin resistant, not Ampicillin resistant. Will reattempt overnight culture on day fifteen.

29/07/10 - Day Fifteen

Selected colonies from the BBa_J04450 containing cells placed in 5mL LB + 1.25μL Kanamycin. Left overnight.

Combined 3mL of BBa_E0840 and BBa_E0240 cell cultures in 1.5mL eppendorf tubes twice - total of 6mL combined cell culture for each biobrick. Performed a miniprep on the combined cultures using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 100μL of solution containing BBa_E0840 and BBa_E0240 biobricks in high concentration.

Restreaked colonies from the XL1-Blue cells containing BBa_K216009 biobrick. Left overnight.

30/07/10 - Day Sixteen

Combined 3mL of BBa_J04450 cell cultures in 1.5mL eppendorf tubes twice - total of 6mL combined cell culture for each biobrick. Performed a miniprep on the combined cultures using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 100μL of solution containing BBa_J04450 biobrick at a high concentration.

Transformed competent E.coli strain M182 with BBa_K216009. Plated out on Ampicillin plates along with a negative control in preparation for a Miller Assay. Also transformed MG1655s with BBa_I13522 (AmpR) and BBa_J04450 (KanR) in preparation for experiments in soil with mixed cultures. Left over weekend at 30°C.

02/08/10 - Day Seventeen

Restreaked M182s + BBa_K216009 on Agar + Amp plates. Left overnight.

(+ other team member's write up to follow)

03/08/10 - Day Eighteen

Prepared overnight cultures of transformed M182s. Prepared buffers and solutions for β-galactosidase assay. Full details to follow.

(+ other team member's write up to follow)

04/08/10 - Day Nineteen

Performed β-galactosidase assay. Details to follow. No activity observed.

(+ other team member's write up to follow)

05/08/10 - Day Twenty

Performed double digest on BBa_K216009 provided by registry, to check identity of the biobrick. Results pending.

(+ other team member's write up to follow)

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.