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From 2010.igem.org

Notebook

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 [http://partsregistry.org/Part:BBa_I13522 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 [http://partsregistry.org/Part:BBa_I13522 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 [http://partsregistry.org/Part:BBa_I13522 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 + [http://partsregistry.org/Part:BBa_I13522 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 [http://partsregistry.org/Part:BBa_K216005 BBa_K216005] and the other containing [http://partsregistry.org/Part:BBa_K216009 BBa_K216009]. Left overnight.

27/07/2010 - Day Thirteen

Selected colonies from the plates of E.coli containing the biobricks [http://partsregistry.org/Part:BBa_K216005 BBa_K216005] and [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] placed in 50mL LB + 50μL Ampicillin. Left overnight.

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

28/07/10 - Day Fourteen

Combined 3mL of [http://partsregistry.org/Part:BBa_K216005 BBa_K216005] and [http://partsregistry.org/Part:BBa_K216009 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 [http://partsregistry.org/Part:BBa_K216005 BBa_K216005] and [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] biobricks in high concentration.

Performed transformation of XL1-Blue E.coli cells with 2μL eluted [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] solution per 100μL cells. Left overnight.

Selected colonies from the Nova blue E.coli plates with the biobricks [http://partsregistry.org/Part:BBa_E0840 BBa_E0840], [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] and [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] were taken and placed in 5mL LB + 5μL Ampicillin. Left overnight. Writing this, realised that the cells containing the [http://partsregistry.org/Part:BBa_J04450 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 [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] containing cells placed in 5mL LB + 1.25μL Kanamycin. Left overnight.

Combined 3mL of [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] and [http://partsregistry.org/Part:BBa_E0240 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 [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] and [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] biobricks in high concentration.

Restreaked colonies from the XL1-Blue cells containing [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] biobrick. Left overnight.

30/07/10 - Day Sixteen

Combined 3mL of [http://partsregistry.org/Part:BBa_J04450 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 [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] biobrick at a high concentration.

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

02/08/10 - Day Seventeen

Performed a double digest on 30μL of biobrick [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] with Xba1 and Pst1. Digest was then run on an Agarose gel for 1 hour at 100V then a further hour at 120V resulting in 3 bands; our desired fragment, the vector and the entire plasmid cut only once. DNA of our fragment was then extracted using QIAGEN gel extraction kit and the result frozen for later use.

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

03/08/10 - Day Eighteen

Performed further double digests on [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] using Xba1 and Pst1 and on [http://partsregistry.org/Part:BBa_K216005 BBa_K216005] using Pst1 and Spe1. The [http://partsregistry.org/Part:BBa_K216005 BBa_K216005] digestion was then treated with phosphatase and then run on a gel along with the other digest. DNA from both digests was then extracted using the QIAGEN kit.

Two ligation mixes were then set up using a 1:3 ratio of vector (the [http://partsregistry.org/Part:BBa_K216005 BBa_K216005] digest) to insert ( [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] and [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] ). A further ligation was set up without any insert to give us an idea of how many times the vector will religate without any insert.

These ligation mixes were then put on ice and left overnight, with the ice allowed to melt causing a gradual temperature rise.

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

04/08/10 - Day Nineteen

Transformed commercial NovaBlue cells with our new biobricks. 30μL of cells were mixed with 5μL of each ligation mix plus 250μL of SOC medium. 100μL of each mix was plated on Ampicillin plates, resulting in three plates, two containing new biobricks and one just the vector . The remaining mixes were then centrifuged, and the pellet resuspended in approximatly 100μL of supernatent. These were then also plated and all plates incubated overnight at 37°C.

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

05/08/10 - Day Twenty

Checked transformations from day before and set up two overnights with colonies picked from the promotor - [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] ligation plate, plus two containing colonies picked from the promotor - [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] ligation. Finally, also set up one overnight with no nitrogen, and another containing 20mM of Potassium Nitrate - using a colony containing the promotor - [http://partsregistry.org/Part:BBa_E0840 BBa_E0840] ligation.

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

06/08/10 - Day Twenty One

The overnight containing Potassium Nitrate was visibly greener than the overnight without, suggesting we have successfully created a new biobrick. To confirm this minipreps were performed using 3mL of each of the other overnights in preparation for digestions to check the presence of our inserts.

09/08/10 - Day Twenty Two

Performed a double digest on each of our minipreps using Xba1 and Spe1. These were then run on an agarose gel and viewed under UV. All of the digestions showed the vector at approximately 2kb and our insert at approximately 1kb as expected, indicating in all cases the ligations occured correctly.

10/08/10 - Day Twenty Three

Set up overnights of MG1655s transformed with our two new biobricks in varying concentrations of Potassium Nitrate.

Began our first trials at making beads; the basic procedure involves pipetting dropwise melted Gellan Gum mixed with cell suspension into a salt solution, after experimenting with several different concentrations of both Calcium Chloride and Magnesium Sulphate we settled on the following procedure:

1. Add 0.24g of gellan gum to 19.6mL of ddH2O, heat to 94°C, stirring occasionally, then cool to 54°C . 2. Centrifuge 2 x 25mL of overnight at 4500 x g for 8 mins and discard supernatent. 3. Wash pellet in 2mL of ddH2O, resuspend and centrifuge for 5 mins at 4500 x g. Repeat once. 4. Slurry cells in 0.25mL of ddH2O and add to cooled gum, mix gently. 5. Pipette dropwise into 0.5M of Calcium Chloride using the P1000. 6. Gently agitate then pour off the salt solution.

This procedure yields beads approximately 2mm in diameter, which in our initial trials using overnights of constitutive GFP production were bright green.

11/08/10 - Day Twenty Four

First observed overnights of varying KNO3 concentrations. Whilst there was a visible difference between no nitrogen and 10mM KNO3 it was impossible to distinguish by eye between the overnights of 10 - 100mM KNO3

Also set up a 50mL overnight of MG1655 plus the new biobrick (strong RBS) in preparation for making new beads.

Finally calculated how much DNA each of the minipreps from 06/08 yielded in preparation for sending these for sequencing.

12/08/10 - Day Twenty Five

Following procedure listed on 10/08 made beads containing MG1655 transformed with the strong RBS new biobrick. Some of these were placed in LB broth containing 0, 20 and 50mM of KNO3 and left overnight.

Glycerol stock of MG1655 plus the strong RBS new biobrick were made and frozen at -80°C.

XL1 blues were transformed with this new biobrick in preparation for performing a midiprep.

13/08/10 - Day Twenty Six

Observed the new beads under UV light. There was a small but visible difference in florescence between the nitrogen and no nitrogen beads, our next step is to get a quantifiable measurement of this difference.

16/08/10 - Day Twenty Seven

Samples of our new biobricks were sent off to the University of Dundee for sequencing. 6μL of plasmid diluted in 54μL of ddH2O were sent off of both the strong and weak RBS new biobricks. The primers sent were the VR reverse primer and SB1A3 forward primer diluted at 0.5μL of primer in 15μL of ddH2O for each. These primers were obtained from last year's stock.

An overnight of XL1 blues transformed with the new biobrick was set up in preparation for a midiprep.