Team:Cambridge/Bioluminescence/Vibrio Characterisation

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{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}

This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases ''lux operon'' from ''Vibrio fischeri'']. 

*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Description|Description]]
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Arabinose_to_light|Arabinose to light]]
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#H-NS_mutants|H-NS mutants]]



=Description=
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter. D-Luciferin has to be added to obtain light output.

=Arabinose to light=
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. 

{{:Team:Cambridge/Templates/Nolineheader|header=Data}}

'''Transfer function of <partinfo>K325219</partinfo>.'''
 
[http://partsregistry.org/wiki/images/2/26/IntegratedactiPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]

The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. 

'''Maximum luminescence output of  <partinfo>K325219</partinfo> as a function of Arabinose concentration.'''

[http://partsregistry.org/wiki/images/0/05/MaximumlumPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/0/05/MaximumlumPP%2BLRE.png/569px-MaximumlumPP%2BLRE.png]


These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.

'''Evolution of luminescence with time at different Arabinose concentrations.'''

[http://partsregistry.org/wiki/images/4/4c/TimePP%2BLRE.png http://partsregistry.org/wiki/images/thumb/4/4c/TimePP%2BLRE.png/569px-TimePP%2BLRE.png]

The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.
<center>
{|{{Table}}
!Data
!Notes
!Date Uploaded
|-
|[http://partsregistry.org/wiki/images/8/82/BBa_K325219ArabinosetoLight.xls Media:BBa_K325219ArabinosetoLight.xls]
|Raw data from experiment
|21/10/2010
|}
</center>


{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}

=Luciferin to light=

The light output is also a function of the concentration of D-Luciferin the media. This page describes the relationship between D-Luciferin concentration and light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. 

{{:Team:Cambridge/Templates/Nolineheader|header=Data}}

'''Effect of D-Luciferin concentration on light output from <partinfo>K325219</partinfo>.'''

[http://partsregistry.org/wiki/images/c/c3/Luciferineffect.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]

The data points represent the mean of 31 values obtained for light output at 30 min interval from 1500 min to 2400 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 31 data points centred around the mean value.

'''Evolution of luminescence with time at different Arabinose concentrations.'''

[http://partsregistry.org/wiki/images/b/b9/Luctimecourse.png http://partsregistry.org/wiki/images/thumb/b/b9/Luctimecourse.png/569px-Luctimecourse.png]

The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.
<center>
{|{{Table}}
!Data
!Notes
!Date Uploaded
|-
|[http://partsregistry.org/wiki/images/9/9a/BBa_K325219luciferintolight.xls Media:BBa_K325219luciferintolight.xls‎]
|Raw data from experiment
|21/10/2010
|}
</center>

{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}

#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>.  One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before.  This growth took on average 4.5 hrs.
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). 
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M).  Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL].  Three wells were each filled with 200 µl of medium to measure the absorbance background.  Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds).  Time between repeated measurements was 2 min and 21 s.  Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement.  [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading.  Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate.  
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page].  The data for each colony tested was averaged across the three replicate wells.  The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3.  Following an initial transient response, device output reached an approximate steady state. 
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line).  Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples.  The cyan shaded region represents the range of the nine independent samples.
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data.  A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99).  In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above.  Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data.  The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.

=Effect of pH=

Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. 

{{:Team:Cambridge/Templates/Nolineheader|header=Data}}

'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''

[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]

These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.

'''Evolution of light output at different values of pH.'''

[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]

Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.
<center>
{|{{Table}}
!Data
!Notes
!Date Uploaded
|-
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]
|Raw data from experiment
|21/10/2010
|}
</center>

{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].

=Compatibility=
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br>

=References=
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.


[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.


[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.


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