Team:Kyoto/Project/Goal A

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==Goal A: Characterization of R0011, a strong lactose promoter==
==Goal A: Characterization of R0011, a strong lactose promoter==
===Introduction===
===Introduction===
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We used <partinfo>R0011</partinfo> [[Team:Kyoto/LearnMore#R0011|[learn more]]], a lactose promoter, in the experiment of lysis box.
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We used <partinfo>R0011</partinfo> [[Team:Kyoto/LearnMore#R0011|[learn more]]], a lactose promoter, for the lysis box.
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Because part parameters of R0011 are partially uncertain <sup>[[#RefA001|[1]]]</sup>, we characterized R0011 in order to characterize lysis cassette.
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Because part parameters of R0011 are partially unclear <sup>[[#RefA001|[1]]]</sup>, we measured the promoter activity of the R0011 in order to characterize lysis cassette.
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We characterize R0011 by Relative Promoter Unit (RPU) [[Team:Kyoto/LearnMore#Relative Promoter Unit (RPU)|[learn more]]]. When absolute promoter activity is measured at various test condition and measurement instruments, there is variation in promoter activity; RPU can reduce this Coefficient of variation (CV) from 39.1% to17.5% <sup>[[#RefA002|[2]]]</sup>. Therefore RPU can make it easier for us to share the data of promoter activity and use biobricks.
+
We characterize R0011 by Relative Promoter Unit (RPU) [[Team:Kyoto/LearnMore#Relative Promoter Unit (RPU)|[learn more]]]. [learn more], bacause absolute promoter activity depends on test conditions and measurement instruments. RPU can reduce this Coefficient of variation (CV) from 39.1% to17.5% <sup>[[#RefA002|[2]]]</sup>. Therefore RPU can make it easier for us to share the data of promoter activity and use biobricks.
===Method===
===Method===

Revision as of 21:16, 27 October 2010

Contents

Goal A: Characterization of R0011, a strong lactose promoter

Introduction

We used BBa_R0011 [learn more], a lactose promoter, for the lysis box.

Because part parameters of R0011 are partially unclear [1], we measured the promoter activity of the R0011 in order to characterize lysis cassette.

We characterize R0011 by Relative Promoter Unit (RPU) [learn more]. [learn more], bacause absolute promoter activity depends on test conditions and measurement instruments. RPU can reduce this Coefficient of variation (CV) from 39.1% to17.5% [2]. Therefore RPU can make it easier for us to share the data of promoter activity and use biobricks.

Method

In order to measure RPU of R0011, we use three types of E.coli, E.coli KRX transformed with BBa_K358000, KRX transformed with BBa_K358001, KRX transformed with pSB4K5 without insert.

The reason we use KRX, one of E.coli strains is, that lacI repressor is over expressed in KRX and lacI can repress lactose promoter fully at low IPTG concentration [3].

KyotoFigA001.png
KyotoFigA002.png

KyotoFigA003.png

KyotoFigA004.png

In order to measure RPU, we must use two types of E.coli transformed with the test promoter circuit and the standard promoter circuit respectively to compare absolute activity of test promoter with that of the standard. BBa_K358000 is the lactose promoter, R0011 with GFP and BBa_K358001 is the standard promoter with GFP. Both parts are combined with pSB4K5, a low copy plasmid, because lacI cannot repress the actiivty of lactose promoter enough with low IPTG concentration and too much lactose promoter DNA. The E.coli transformed with pSB4K5 without the insert is also used to correct the autofluorescence.

We pick up three colonies from each plate, and cultivate them in the supplemented M9 medium for a night, about 16 hours. The overnight cultures are diluted to 1:100 in pre-warmed fresh the supplemented M9 medium. 3h later and 3.5h later, we measure OD600 of the culture and GFP fluorescence in the culture. We firstly measure RPU after cells are lyzed. The measurement of RPU with some IPTG is done without cell lysis.

To know more detail procedures, go Protocols and Notebook.

We measure OD every 30 minutes after dilution to ensure that cells in log phase growth.

RPU is calculated as follows.

KyotoGoalA001.png

We also use these rough calculations;

KyotoGoalA002.png
KyotoGoalA003.png

Here, F3h and F3.5h is the GFP fluorescence measured at 3h and 3.5h respectively,

ABS3h and ABS3.5h is OD600 of the culture at 3h and 3.5h respectively.

From the experimental data for cell growth from 2h to 4h after dilution, we thought cell growth from 3h to 3.5h after dilution can be approximated as linear. If GFP concentration in cell is in steady states, the increase of GFP fluorescence depends on only cell growth. We used the first approximation based on such assumptions. We also used the second based on that cell growth from 3h to 3.5h after dilution can be approximated as linear and the mean ABS can be calculated by using this equation.

Result

The result of characterization is shown in table and figure below.

IPTG (mM)RPU
ABCAverageStdevCV
0*0.01220.01470.01340.008210.611
0.010.01910.009630.002440.01040.00840.806
0.030.1770.1550.1980.1740.01730.0997
0.10.2050.1910.2690.2210.04160.188
0.30.9380.9550.9550.9490.009810.0103
0.81.44*1.211.370.1610.122
1.01.601.581.651.650.1060.0640
2.01.491.831.391.570.2330.148
  • *One of RPU in IPTG 0mM couldn’t be measured. One of RPU in IPTG 0.8mM couldn't also be measured.
  • *In this table, RPU in 0, 0.01, 0.1, 1.0 mM IPTG is measured with cell lysis and RPU in 0.05, 0.3, 0.8, 2.0 mM IPTG is measured without cell lysis.

We made the model about the activity of R0011 and decided parameters from the result to estimate RPU of R0011 with IPTG concentrations we couldn’t measure. The result is also drawn in figure below. To know more about this model, go Modelling.

KyotoGrpA003.png
KyotoGrpA001.png

In addition, the result of measurement of cell growth is shown in figure below. These graphs indicate that cells are in log phase growth. These graphs also indicate that cell growth from 3h to 3.5h after dilution can be approximated as linear.

KyotoGrpA002.png

Discussion

The result indicates that the maximum activity of R0011 is about 1.6 RPU and the minimum is less than 0.01 RPU and that R0011 has wide range of activity ( maximum activity of R0011 is about 160 fold greater than the minimum activity at least). The maximum activity of R0011, 1.6 RPU agrees with the previous data [4]. The wide range of activity of RPU can change the effect of lysis cassette variously, and it is useful to characterize lysis cassette.

The graph of cell growth indicates that cells in log phase growth at 3h and 3.5h after dilution. This means that time of measurement for RPU is correct. In this characterization, there is two way to measure RPU, one is measure GFP fluorescence after cell lysed and the other is measure GFP fluorescence without cell lysed. We think both measurements is valid because RPU of R0011 with 1mM IPTG measured with cell lysis corresponds to the previous data [4] and the way of measurement of RPU without cell lysed is recommended one by the previous igem team [5].

By the way, found that there is a little lactose, approximately 0.01mM lactose, in the supplemented M9 medium.

KyotoGrp100915-1.png

We thought that lactose in the supplemented M9 medium comes from casamino acids because casamino acids made from casein. Therefore, we tried to culture E.coli and measure RPU without the casamino acids because lactose in the supplemented M9 medium can induce R0011. However, without casamino acids, the growth rate of E.coli declines and we could not measure RPU. The all RPU data was measured with casamino acids. Accordingly, the real value for RPU of R0011 at low IPTG concentration may be lower than the value gained from this experiment. Measurement of lactose concentration is done by using Lactose Assay Kit (BioVison) .[6] CV in 0mM IPTG is so high, 0.611, and CV in 0.01mM IPTG is also high, 0.806. We cannot trust RPU in 0mM and 0.01mM ITPG so much.

Finally, we could characterize R0011 at high IPTG concentration correctly. However, characterization of R0011 at low IPTG concentration may be required additional measurements.

Reference

  1. http://partsregistry.org/Part:BBa_R0011
  2. Jason R Kelly et al. ”Measuring the activity of BioBrick promoters using an in vivo reference standard.” Journal of Biological Engineering 2009, 3:4
  3. http://www.promega.com/pnotes/94/14410_27/14410_27.pdf
  4. http://partsregistry.org/Part:BBa_R0011:Experience
  5. http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Fluorescence#Fluorescence_and_water_dispensation
  6. http://www.biovision.com/manuals/K624-100.pdf