Revision as of 03:36, 21 September 2011

Characterisation

Our CryoBricks are designed to facilitate cold resistance in E. coli. We characterise this cold resistance in two different ways.

The first characterisation method is measuring freeze/thaw cycle survival rate. We exposed E. coli cultures to subsequent freezing and thawing. After each thaw step, part of the culture was plated out, and after ~12 hours at 37°C, colony forming units (CFUs) were counted on the plates. These CFUs represent the number of viable cells present in the culture. Cold resistant cells will have a higher fraction of cells surviving each cycle. The protocol followed for these experiments can be found on this page.

The second method through which we characterise cold resistance is more relevant to most of our foreseen applications. It involves measuring the effect of expressing a CryoBrick on the specific growth rate (μ) of an organism, at temperatures suboptimal for growth. For normal E. coli, μ peaks at 37°C, making any temperature lower than this suboptimal. The rate at which μ declines when temperatures drop below this optimum is an indication of cold resistance, as more cold resistant E. coli will be able to maintain a higher μ at low temperatures.

Because μ is defined as the increase of biomass per unit of time, per unit of biomass, it can be estimated from time-series data of a logistically growing population's density. To characterise the effect of CryoBrick expression on growth at suboptimal temperatures, we culture E. coli comprising a CryoBrick at different temperatures, using the culture's optical density at a wavelength of 600nm (OD600) as an indicator of population density, and measuring this at frequent time intervals during culture growth. The protocol used to obtain this data can be found here. How we subsequently analyse the gathered data to estimate μ is explained on the data analysis page.

Results

At the time of this writing, two of the designed CryoBricks have been characterised, with both characterisation methods. The first is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538000 BBa_K538000], encoding Cpn10. It was expressed in E. coli through part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538200 BBa_K538200]: a protein generator under control of the Lac operon's promoter, incorporating one of the stronger Community Collection RBSes ([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034]). The generator was transformed into the Top10 strain, which is LacI deficient. As such, the part should be expressed constitutively, and no IPTG or lactose is needed to activate expression. Note that Cpn10 has only ever been reported to be functional when coexpressed with its partner protein Cpn60, so we expected it not to have any influence on cold resistance at all.

The second CryoBrick we've characterised succesfully is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538004 BBa_K538004]: Polaribacter irgensii 's CspC protein, expressed in our bacteria through [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538204 BBa_K538204], which only differs from the Cpn10 generator in that it contains the CspC coding region instead. This protein has been reported to strongly increase the freeze/thaw cycle survival rate of E. coli, so we expected to reproduce this observation. We had no particularly strong prior expectations about this enzyme influencing growth rate at suboptimal temperatures, as nothing of the sort has been reported in literature to date.

Freeze/Thaw Cycle Survival

[Update pending]

Growth at Suboptimal Temperatures

The growth of 4 different strains was measured at 7 different temperatures. A wildtype strain, a strain comprising an empty vector (with an antibiotic resistance, but no CryoBrick coding regions), and strains comprising either the Cpn10 generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K538200 BBa_K538200]) or the CspC generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K538204 BBa_K538204]) were cultured at 4, 8, 15, 21, 25, 30 and 37°C. If expression of a CryoBrick influences growth rate at any of these temperatures, we expect the effect to be the strongest at the lowest temperatures. Conversely, as comprising the plasmids shouldn't hinder the cells in optimal circumstances, no differences are expected at 37°C. The results of our growth rate characterisation experiments are displayed in figure 2, below.

Figure 2: E. coli 's growth rate as a function of temperature, in strains that do or do not express CryoBricks. The amount of measured cultures an average and its standard deviation are based is displayed above each bar. Error bars indicate the average plus or minus two standard deviations. Click to view in a different screen.

Conclusion

Figure 2 shows that, according to expectations, expression of Cpn10 does not significantly influence growth rates at suboptimal temperatures, at least in the concentration at which it's expressed through this construct. No a priori expectations existed regarding CpsC 's influence on these growth rates, but apparently, expression of this CryoBrick doesn't allow E. coli to maintain higher growth rates at suboptimal temperatures either.

We have, however, confirmed that the CspC CryoBrick is functional and that it influences the freeze/thaw cycle survival of E. coli as we expected it to based on literature. Also of particular interest is that expression of Cpn10 alone can enhance cold resistance, and even though it's not as efficient as CspC when it comes to increasing freeze/thaw survival, this is to the best of our knowledge the first time Cpn10 has been demonstrated to significantly affect E. coli independently of its partner protein Cpn60!

@@ Line 1: / Line 1: @@
 {{:Team:Amsterdam/Header}}
-=Characterization=
+__NOTOC__
+=Characterisation=
+Our CryoBricks are designed to facilitate cold resistance in ''E. coli''. We characterise this cold resistance in two different ways.
+<br><br>
+The first characterisation method is measuring freeze/thaw cycle survival rate. We exposed ''E. coli'' cultures to subsequent freezing and thawing. After each thaw step, part of the culture was plated out, and after ~12 hours at 37°C, colony forming units (CFUs) were counted on the plates. These CFUs represent the number of viable cells present in the culture. Cold resistant cells will have a higher fraction of cells surviving each cycle. The protocol followed for these experiments can be found on [[Team:Amsterdam/Notebook/Protocols/Freeze_thaw|this]] page.
+<br><br>
+The second method through which we characterise cold resistance is more relevant to most of our foreseen [[Team:Amsterdam/Project/Applications|applications]]. It involves measuring the effect of expressing a CryoBrick on the specific growth rate (μ) of an organism, at temperatures suboptimal for growth. For normal ''E. coli'', μ peaks at 37°C, making any temperature lower than this suboptimal. The rate at which μ declines when temperatures drop below this optimum is an indication of cold resistance, as more cold resistant ''E. coli'' will be able to maintain a higher μ at low temperatures.
+<br><br>
+Because μ is defined as ''the increase of biomass per unit of time, per unit of biomass'', it can be estimated from time-series data of a logistically growing population's density. To characterise the effect of CryoBrick expression on growth at suboptimal temperatures, we culture ''E. coli'' comprising a CryoBrick at different temperatures, using the culture's optical density at a wavelength of 600nm (OD600) as an indicator of population density, and measuring this at frequent time intervals during culture growth. The protocol used to obtain this data can be found [[Team:Amsterdam/Notebook/Protocols/Growth_Curve|here]]. How we subsequently analyse the gathered data to estimate μ is explained on the [[Team:Amsterdam/Labwork/Data_Analysis|data analysis]] page.
-So in the end, we got two working assembly’s.
+==Results==
+At the time of this writing, two of the designed CryoBricks have been characterised, with both characterisation methods. The first is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538000 BBa_K538000], encoding ''Cpn10''. It was expressed in ''E. coli'' through part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538200 BBa_K538200]: a protein generator under control of the Lac operon's promoter, incorporating one of the stronger Community Collection RBSes ([http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034]). The generator was transformed into the Top10 strain, which is ''LacI'' deficient. As such, the part should be expressed constitutively, and no IPTG or lactose is needed to activate expression. Note that ''Cpn10'' has only ever been reported to be functional when coexpressed with its partner protein ''Cpn60'', so we expected it not to have any influence on cold resistance at all.
-==Cpn10==
+<br><br>
-The first is a construct with lacI, strong RBS and Cpn10. In our E.coli lacI is always present so the promoter is always on. Cpn10 was selected, because it is a chaperone that’s works together with Cpn60 and helps the folding of proteins at lower temperatures.
+The second CryoBrick we've characterised succesfully is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538004 BBa_K538004]: ''Polaribacter irgensii'' 's ''CspC'' protein, expressed in our bacteria through [http://partsregistry.org/wiki/index.php?title=Part:BBa_K538204 BBa_K538204], which only differs from the ''Cpn10'' generator in that it contains the ''CspC'' coding region instead. This protein has been reported to strongly increase the freeze/thaw cycle survival rate of ''E. coli'', so we expected to reproduce this observation. We had no particularly strong prior expectations about this enzyme influencing growth rate at suboptimal temperatures, as nothing of the sort has been reported in literature to date.
-We do not expect any influence of Cpn10 on the growth curves, because it is the smaller one of the two chaperones. Unfortunately we didn’t get Cpn60 in a construct.
+<br><br>
+===Freeze/Thaw Cycle Survival===
-==CspC==
+[Update pending]
-The second construct is lacI, strong RBS and CspC. CspC is known for its positive influence on survival rate in a Freeze/thaw cycle. There is no indication that CspC has an effect on the growth rate at lower temperatures.
+<br><br>
+===Growth at Suboptimal Temperatures===
-==Growth curves==
+The growth of 4 different strains was measured at 7 different temperatures. A wildtype strain, a strain comprising an empty vector (with an antibiotic resistance, but no CryoBrick coding regions), and strains comprising either the ''Cpn10'' generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K538200 BBa_K538200]) or the ''CspC'' generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K538204 BBa_K538204]) were cultured at 4, 8, 15, 21, 25, 30 and 37°C. If expression of a CryoBrick influences growth rate at any of these temperatures, we expect the effect to be the strongest at the lowest temperatures. Conversely, as comprising the plasmids shouldn't hinder the cells in optimal circumstances, no differences are expected at 37°C. The results of our growth rate characterisation experiments are displayed in figure 2, below.
-[under construction]
+<br><br>
+<!-- Size specification parsing malfunctions! -->
+[[Image:Growth_rate_chart.png|center|frame|Figure 2: ''E. coli'' 's growth rate as a function of temperature, in strains that do or do not express CryoBricks. The amount of measured cultures an average and its standard deviation are based is displayed above each bar. Error bars indicate the average plus or minus two standard deviations. '''Click to view in a different screen.''']]
-We expected that if our genes would have any effect, it would be a higher specific growth rate at low temperatures (4 to 30°C), and no effect on the specific growth rate at 37°C (the optimal temperature for E.coli). Figure 1 shows that Cpn10 and CspC do not contribute to a higher specific growth rate at lower temperatures.
+==Conclusion==
+Figure 2 shows that, according to expectations, expression of ''Cpn10'' does '''not''' significantly influence growth rates at suboptimal temperatures, at least in the concentration at which it's expressed through this construct. No a priori expectations existed regarding ''CpsC'' 's influence on these growth rates, but apparently, expression of this CryoBrick doesn't allow ''E. coli'' to maintain higher growth rates at suboptimal temperatures either.
+<br><br>
+We have, however, confirmed that the ''CspC'' CryoBrick is functional and that it influences the freeze/thaw cycle survival of ''E. coli'' as we expected it to based on literature. Also of particular interest is that expression of ''Cpn10'' alone '''can''' enhance cold resistance, and even though it's not as efficient as ''CspC'' when it comes to increasing freeze/thaw survival, this is to the best of our knowledge the first time ''Cpn10'' has been demonstrated to significantly affect ''E. coli'' independently of its partner protein ''Cpn60''!
 {{:Team:Amsterdam/Footer}}

Team:Amsterdam/Labwork/Characterization

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