Team:BCCS-Bristol/Wetlab/Notebook

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(Wet lab)
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== Wet lab ==
== Wet lab ==
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For a complete list of the biobricks used during our project, plus a brief description on their use, click [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/BioBricks here].
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'''09/07/2010 - Day One'''
'''09/07/2010 - Day One'''

Revision as of 15:20, 27 July 2010

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 - 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 2010 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 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

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 broth + 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.