Team:Edinburgh/Project/Results/Chassis

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<h3>Characterisation of ''Bacillus subtilis 168'' as a chassis</h3>
<h3>Characterisation of ''Bacillus subtilis 168'' as a chassis</h3>
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In order to better anticipate what conditions are best suitable for growth of our experimental strain, <i> Bacillus subtilis</i> 168, we tested the growth under various conditions relevant to the work we are going to perform. First we measured the growth curves at various ethanol concentrations to confirm the highest ethanol concentration suitable for cell growth, which was 4%, displayed bellow. Afterwards, we measured the growth curves at various concentrations of iron, copper, nickel, cobalt, zinc and manganese, displayed bellow.
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In order to better understand what conditions are best suitable for growth of our experimental strain, <i> Bacillus subtilis</i> 168, we tested the growth under various conditions relevant to the work we would perform later. First we measured the growth curves at various ethanol concentrations to confirm the highest ethanol concentration suitable for cell growth, which was 4%, as displayed below. Afterwards, we measured the growth curves at various concentrations of iron, copper, nickel, cobalt, zinc and manganese, which are also displayed below.
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<h3>Testing the Kanamycin resistance conferred by pTG262 (BBa_I742123) in B.subtilis and E.coli</h3>
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<div id="vector"><h3>Testing the Kanamycin resistance conferred by pTG262 (BBa_I742123) in <i>B.subtilis</i> and <i>E.coli</i></h3></div>
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B.subtilis 168 and E. coli JM109 were each transformed with the vector pTG262 (BBa_I742123) with an RFP biobrick insert cassette.
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<i>B.subtilis</i> 168 and <i>E. coli</i> JM109 were each transformed with the vector pTG262 (BBa_I742123) with an RFP biobrick insert cassette.
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<h2>B.subtilis</h2>
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<h2><i>B.subtilis</i></h2>
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WT B. ubtilis 168 and pTG262 transformed B.subtilis 168 colonies were used to inoculate 5ml LB medium in glass standard bottles which were incubated at 37C with shaking and loose lids for  ~6h (pTG262 with 5mg.L-1 chloramphenicol to prevent vector loss). This culture was then diluted by a factor of 10-6 and 100ul aliquots were plated onto LB agar plates of varying kanamycin concentrations, and incubated overnight at 37C.
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Wild type ''B. subtilis'' 168 and pTG262 transformed ''B.subtilis'' 168 colonies were used to inoculate 5 ml of LB medium intp glass standard bottles which were incubated at 37° C with shaking for  ~6h (pTG262 with 5 mg.L-1 chloramphenicol to prevent vector loss). This culture was then diluted by a factor of 10:6 and 100ul aliquots were plated onto LB agar plates of varying kanamycin concentrations, and subsequently incubated overnight at 37° C.
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The number and size of colonies which grew on each of the agar plates was recorded.
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The number and size of colonies which grew on each of the agar plates was recorded as seen below.
[[File:B.subAgar.jpg]]  
[[File:B.subAgar.jpg]]  
   
   
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<h2>E.coli test of growth on agar plates</h2>
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<h2><i>E.coli</i> test of growth on agar plates</h2>
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WT E.coli JM109 and pTG262 transformed E.coli JM109 colonies were used to inoculate 5ml LB medium in glass standard bottles which were incubated at 37C with shaking for 6h30m (pTG262 inoculated in duplicate with no antibiotic and 10mg.L-1 Kanamycin).  
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Wild type <i>E.coli</i> JM109 and pTG262 transformed <i>E.coli</i> JM109 colonies were used to inoculate 5 ml LB medium in glass standard bottles which were incubated at 37° C with shaking for 6 hours and 30 minutes (pTG262 inoculated in duplicate with no antibiotic and 10 mg.L-1 Kanamycin).  
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The OD600 of the cultures was measured:
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The OD600 of the cultures was measured as shown below:
[[File:E.coliInoculumforplates.jpg]]
[[File:E.coliInoculumforplates.jpg]]
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Each of the above cultures were diluted by factors of 10-5 and 10-6. Agar plates of varying kanamycin concentration were plated with 100ul aliquots of the dilutions, and incubated for ~36h at 37C. The number and colour of colonies was recorded (red indicating presence of pTG262, white being WT or representing loss of the plasmid before plating).
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Each of the above cultures were diluted by factors of 10:5 and 10:6. Agar plates of varying kanamycin concentration were plated with 100 μl aliquots of the dilutions, and incubated for ~36 hr at 37° C (Figure 1). The number and colour of the colonies were recorded (red indicating presence of pTG262, white being wild type or representing loss of the plasmid before plating).
   
   
[[File:E.coliAgar.jpg]]
[[File:E.coliAgar.jpg]]
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[[File:E.coliAgarpicture.jpg|600px|]]
[[File:E.coliAgarpicture.jpg|600px|]]
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Figure 1. A plate from the above experiment after an additional 24 hours on the bench, pTG262 Kan0 10-5 dilution plated onto 0mg.L-1 Kanamycin. This shows the typical colony size as indicated in the above table. It demonstrates both presumed plasmid loss in overnight culture without antibiotic (white colonies) and the decreased growth rate of cells containing pTG262.
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Figure 1. A plate from the above experiment after an additional 24 hr on the bench, pTG262 Kan0 10:5 dilution plated onto 0 mg.L-1 Kanamycin. This shows the typical colony size as indicated in the above table. It demonstrates both presumed plasmid loss in overnight culture without antibiotic (white colonies) and the decreased growth rate of cells containing pTG262.
   
   
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<h2>E. coli test of growth in Kanamycin liquid culture</h2>
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<h2><i>E. coli</i> test of growth in Kanamycin liquid culture</h2>
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Overnight LB liquid cultures of WT E.coli JM109 and E.coli JM109 transformed with pTG262 were diluted 1:50 in fresh LB medium. 3ml aliquots of each were put in glass standard bottles, along with varying amounts of kanamycin. The Optical density at 600nm was measured at the start and after 8 hours incubation with shaking at 37C.
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Overnight LB liquid cultures of wild type <i>E.coli</i> JM109 and <i>E.coli</i> JM109 transformed with pTG262 were diluted 1:50 in fresh LB medium. 3 ml aliquots of each were put in glass standard bottles, along with varying amounts of kanamycin. The Optical density at 600 nm was measured at the start and after 8 hr incubation with shaking at 37° C.
Initial OD600: WT JM109 - 0.030, pTG262 - 0.028.
Initial OD600: WT JM109 - 0.030, pTG262 - 0.028.
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OD600 after 8 hours incubation with shaking at 37C:
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OD600 after 8 hr incubation with shaking at 37° C:
[[File:LiquidcultureE.coli.jpg]]
[[File:LiquidcultureE.coli.jpg]]

Latest revision as of 23:14, 4 October 2013

Contents

Characterisation of Bacillus subtilis 168 as a chassis

In order to better understand what conditions are best suitable for growth of our experimental strain, Bacillus subtilis 168, we tested the growth under various conditions relevant to the work we would perform later. First we measured the growth curves at various ethanol concentrations to confirm the highest ethanol concentration suitable for cell growth, which was 4%, as displayed below. Afterwards, we measured the growth curves at various concentrations of iron, copper, nickel, cobalt, zinc and manganese, which are also displayed below.


EdiChassis.png
EdiChassis1.png
EdiChassis2.png
EdiChassis3.png
EdiChassis4.png
EdiChassis5.png
EdiChassis6.png


Testing the Kanamycin resistance conferred by pTG262 (BBa_I742123) in B.subtilis and E.coli

B.subtilis 168 and E. coli JM109 were each transformed with the vector pTG262 (BBa_I742123) with an RFP biobrick insert cassette.

B.subtilis

Wild type B. subtilis 168 and pTG262 transformed B.subtilis 168 colonies were used to inoculate 5 ml of LB medium intp glass standard bottles which were incubated at 37° C with shaking for ~6h (pTG262 with 5 mg.L-1 chloramphenicol to prevent vector loss). This culture was then diluted by a factor of 10:6 and 100ul aliquots were plated onto LB agar plates of varying kanamycin concentrations, and subsequently incubated overnight at 37° C.

The number and size of colonies which grew on each of the agar plates was recorded as seen below.

B.subAgar.jpg

E.coli test of growth on agar plates

Wild type E.coli JM109 and pTG262 transformed E.coli JM109 colonies were used to inoculate 5 ml LB medium in glass standard bottles which were incubated at 37° C with shaking for 6 hours and 30 minutes (pTG262 inoculated in duplicate with no antibiotic and 10 mg.L-1 Kanamycin).

The OD600 of the cultures was measured as shown below:

E.coliInoculumforplates.jpg

Each of the above cultures were diluted by factors of 10:5 and 10:6. Agar plates of varying kanamycin concentration were plated with 100 μl aliquots of the dilutions, and incubated for ~36 hr at 37° C (Figure 1). The number and colour of the colonies were recorded (red indicating presence of pTG262, white being wild type or representing loss of the plasmid before plating).

E.coliAgar.jpg

E.coliAgarpicture.jpg

Figure 1. A plate from the above experiment after an additional 24 hr on the bench, pTG262 Kan0 10:5 dilution plated onto 0 mg.L-1 Kanamycin. This shows the typical colony size as indicated in the above table. It demonstrates both presumed plasmid loss in overnight culture without antibiotic (white colonies) and the decreased growth rate of cells containing pTG262.

E. coli test of growth in Kanamycin liquid culture

Overnight LB liquid cultures of wild type E.coli JM109 and E.coli JM109 transformed with pTG262 were diluted 1:50 in fresh LB medium. 3 ml aliquots of each were put in glass standard bottles, along with varying amounts of kanamycin. The Optical density at 600 nm was measured at the start and after 8 hr incubation with shaking at 37° C.

Initial OD600: WT JM109 - 0.030, pTG262 - 0.028.

OD600 after 8 hr incubation with shaking at 37° C:

LiquidcultureE.coli.jpg

Liquidculturegraph.jpg