Line 139: | Line 139: | ||
<h7>Rationale and Aim</h7> | <h7>Rationale and Aim</h7> | ||
<!--- Why was this done? what problem is it addressing? How does it address that problem? How does it fit into the rest of the project? What were the aims for this? ---> | <!--- Why was this done? what problem is it addressing? How does it address that problem? How does it fit into the rest of the project? What were the aims for this? ---> | ||
− | + | <p> | |
+ | Previous research has shown that the concentration of some components of the supplement solution are crucial for efficient protein synthesis, and that for each batch of extract produced the optimal concentration may need to be found (Yang, et al., 2012). Studies which have explored this have only focused on, at most, a few components at a time (Garamella, et al., 2016; Kelwick, et al., 2016), which means that important interactions between the components may have been missed. In this study, a multifactorial approach will be taken to investigate the effect that all supplements have on the protein synthesis activity of CFPS systems simultaneously.<br /> | ||
+ | <br /> | ||
+ | The specific aims for this section of the project were: (i) to demonstrate the applicability of | ||
+ | DoE to determine important components of the supplement solution premix, and (ii) to | ||
+ | demonstrate the ability of DoE to predict concentrations of CFPS supplements which yield | ||
+ | optimal protein synthesis activity. | ||
+ | </p> | ||
Line 145: | Line 152: | ||
<h7>Background Information</h7> | <h7>Background Information</h7> | ||
<!--- Reference where the idea came from. Explain the theory behind it. Discuss why it should achieve points given above. ---> | <!--- Reference where the idea came from. Explain the theory behind it. Discuss why it should achieve points given above. ---> | ||
+ | <p> | ||
+ | Traditionally, biologists tend to use One Factor At a Time (OFAT) approaches to determine | ||
+ | the effect and importance of factors on a system, which can sometimes be a poor method. | ||
+ | By only determining the effect that a single factor has on a system at a time, important | ||
+ | interactions can be missed. For example, removing only factor A may have no effect, and | ||
+ | removing only factor B may also have no effect, but removing both may cause an adverse | ||
+ | effect. Therefore, it is important to take a multifactorial approach when investigating the | ||
+ | importance of conditions or components of a system, or when trying to optimise a system. | ||
+ | An issue with this approach is that a large number of experiments may be required to fully | ||
+ | investigate all factors. By using statistical methods, a Design of Experiments (DoE) can be | ||
+ | determined which has the minimum number of experiments required to explore questions | ||
+ | such as the importance of factors in a system. This approach also allows for robustness | ||
+ | testing or determining batch-batch variation (Anderson & Whitcomb, 2010). As discussed | ||
+ | here [Link to Cell Free section of wiki], CFPS systems can be plagued with issues rising from | ||
+ | variation, so this approach offers a method to investigate the causes. It could also be used to | ||
+ | determine less important components of the supplement solution premix which is added to | ||
+ | CFPS systems, and hence a minimal supplement premix could be determined.<br /> | ||
+ | <br /> | ||
+ | There are several different types of DoE designs. One of these is the screening design (SD), | ||
+ | which is used to create experimental designs to determine the factors with the highest effect | ||
+ | on a system. Another design is the surface response design (SRD), which makes | ||
+ | experimental designs to collect data for generating models which can predict optimal | ||
+ | settings for many factors (SAS Institute Inc., 2016). Software tools, such as JMP (SAS | ||
+ | Institute Inc., 2016), can assist in creating these experimental designs. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | <br /> | ||
+ | <h7>Screening Design for Salt Supplements</h7> | ||
+ | <!--- How was this stage designed? What was its sequence (if it's a part)? Was it modelled? What considerations were taken into account when making it? What challenges were there? How does the design link back to the rationale? (i.e. why does this design work). ---> | ||
+ | <p> | ||
+ | |||
+ | Previous research has shown that the concentration of certain salts in the CFPS supplement | ||
+ | premix are crucial for maximal protein synthesis activity [REF]. A Design of Experiments | ||
+ | approach was used to determine which of the four salts (magnesium glutamate, potassium | ||
+ | glutamate, sodium oxalate, and ammonium acetate) are the most important using the JMP | ||
+ | software. A classical screening design was created with all four salts as continuous factors | ||
+ | and CFPS activity as the response to be maximised. A concentration of ‘0’ was used as the | ||
+ | lower limit for each factor, and the concentration used normally in CFPS supplement | ||
+ | premixes was used as the upper limit (Figure 1). The screening design generated is shown | ||
+ | in table 1. | ||
+ | </p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p> | ||
+ | |||
+ | CFPS reactions were performed using | ||
+ | supplement solution premixes with salt | ||
+ | concentrations as determined by the main effects screening design. Reactions were | ||
+ | incubated with 1.7 μg plasmid DNA encoding sfGFP (superfolder Green Fluorescent Protein) | ||
+ | at 37 o C for 13 hours. CFPS activity was calculated as fluorescence intensity at 13 hours | ||
+ | minus fluorescence intensity at 15 mins. This data was then used to generate a bar chart of | ||
+ | Contrast values and a Half-Norma Plot (Figure 2 and 3) to determine which factors were | ||
+ | having the most effect on CFPS activity. It should be noted that predictions for non-primary | ||
+ | factors (i.e. interactions) may be inaccurate as they were forced-orthogonal. Considering the | ||
+ | primary factors, magnesium glutamate was found to be the salt supplement with the largest | ||
+ | contrast value, followed by potassium glutamate. This suggests that these two salt | ||
+ | supplements were the most important. Sodium oxalate had a lower contrast value than | ||
+ | either of the two glutamate salts, and was considered to have moderate importance in terms | ||
+ | of CFPS activity. Ammonium acetate had an extremely low contrast value, suggesting that it | ||
+ | may be unimportant for enhancing CFPS activity. | ||
+ | |||
+ | </p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
Line 150: | Line 243: | ||
<br /> | <br /> | ||
− | <h7> | + | <h7>Surface Response Designs for Salt Supplements</h7> |
− | < | + | <p> |
+ | The DoE software, JMP, was used to create a surface response design (SRD) for the three | ||
+ | salts which were found by the screening design to have the most effect on CFPS activity | ||
+ | (magnesium glutamate, potassium glutamate, and sodium oxalate). Ammonium acetate was | ||
+ | kept at the default concentration and was not varied. Four SRDs were created using JMP; | ||
+ | Central Composite Design-Uniform Precision design (CCD-UP), Box-Behnken (BB), Central | ||
+ | Composite Design-Orthogonal (CCD-O), and Central Composite Design (CCD). The design | ||
+ | diagnostics feature was used to compare the designs (Figure 4). Specifically, the colour map | ||
+ | on correlations, power analysis for each factor and interaction, D, G, and A efficiencies, | ||
+ | average variance of prediction, and number of reactions were compared to determine which | ||
+ | design would be used. The colour map on correlations shows how correlated two terms are | ||
+ | (red is highly correlated, blue is highly un-correlated). The more correlated two terms are, | ||
+ | the more difficult it is to determine which is responsible for the effect on the response | ||
+ | (Anderson & Whitcomb, 2010). As would be expected, in each design, terms are highly | ||
+ | correlated with themselves (observed as a diagonal red line). Other terms are generally very | ||
+ | lowly correlated with different terms. For the CCD-UP, BB, and CCD, the terms at the bottom | ||
+ | right of the map have correlations above 0. For CCD-UP and BB, these correlations are still | ||
+ | very low, but for CCD they are at about 0.5. Power analysis shows the likelihood of detecting | ||
+ | an active effect for terms in the design (Anderson & Whitcomb, 2010). The CCD-O had a | ||
+ | higher Power for all terms, with CCD-UP having the next highest. BB and CCD had lower | ||
+ | Power for all terms, but some terms were higher in the BB design than the CC design, and | ||
+ | some higher in CC design than the BB design. | ||
+ | </p> | ||
+ | <img src=""/> | ||
+ | <p> | ||
+ | |||
+ | D, G, and A efficiencies are a measure of each design to be D, G, and A optimised. A design | ||
+ | is D optimal if confidence regions for the vector of regression coefficients are minimized, G | ||
+ | optimal if maximum prediction variance over the design region is minimized, and A optimal if | ||
+ | the sum of the regression coefficient variance is minimized (Anderson & Whitcomb, 2010). | ||
+ | The CCD-UP design has the highest D efficiency and the BB design has the lowest. The | ||
+ | CCD design has the highest G efficiency and the BB design has the lowest. The CCD-UP | ||
+ | design has the highest A efficiency and the BB design has the lowest.<br /> | ||
+ | <br /> | ||
+ | The last two values analysed to determine which design would be used were the average | ||
+ | variance of prediction, for which CCD-O had the lowest and BB had the highest, and the | ||
+ | number of reactions required by each design, for which CCD-O had the highest and BB had | ||
+ | the lowest. Taking all of the information into account, the CCD-Orthogonal design was | ||
+ | chosen as it has no correlations between non-identical terms, high Power for all terms, | ||
+ | relatively high efficiency scores, and low prediction variation.<br /> | ||
<br /> | <br /> | ||
− | + | CFPS reactions with salt supplement amounts according to the Surface Response | |
− | + | experimental design (Table 2) were performed (Figure 5). Five reactions (12-16) were | |
+ | discarded from analysis. These were all repeats of CFPS systems with default amounts of | ||
+ | salts and showed no CFPS activity due to an error during set-up. Discarding these results | ||
+ | had a minor effect on the diagnostics of the surface response design, with some terms | ||
+ | becoming more correlated (but all values were still below 0.2), the Power for terms | ||
+ | decreasing slightly, and average variance of prediction increasing slightly. Despite this, the | ||
+ | D, G, and A efficiencies all increased. | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td> | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p> | ||
+ | |||
+ | Results for the remaining reactions were used to build a model in JMP to predict an optimal | ||
+ | composition for the three salts. The model predicted that at high amounts, magnesium | ||
+ | glutamate and sodium oxalate were having an inhibitory effect, and potassium glutamate | ||
+ | was having an enhancing effect on CFPS activity (Figure 3.3.3a). It is well known that | ||
+ | magnesium ions are crucial for protein synthesis, for example in the functioning of | ||
+ | ribosomes, however at high amounts magnesium can become inhibiting to protein synthesis | ||
+ | by stalling translation at the translocation step (Li, et al., 2014). Therefore, it is not | ||
+ | unexpected that magnesium glutamate causes a decrease in protein synthesis activity at | ||
+ | certain concentrations.<br /> | ||
<br /> | <br /> | ||
− | + | As mentioned before, sodium oxalate is used as an inhibitor of the enzyme which converts | |
− | + | pyruvate to PEP. However, pyruvate is also con verted to desirable metabolites during the | |
+ | pathway which generates ATP for protein synthesis, and these reactions should not be | ||
+ | inhibited. Sodium oxalate acts as a pyruvate mimic, and therefore at high concentrations it | ||
+ | may not only inhibit conversion of pyruvate to PEP, but also pyruvate to acetyl-CoA, which | ||
+ | would decrease the amount of ATP generate, and hence reduce protein synthesis activity. | ||
+ | This may be one explanation for why sodium pyruvate appears to have an inhibitory affect. | ||
+ | Using this data, a maximum protein synthesis activity within the range of concentrations | ||
+ | used for each salt was found at 6 mM magnesium glutamate, 195 mM potassium glutamate, | ||
+ | and 2 mM sodium oxalate (figure 3.3.3a). A CFPS supplement solution with these revised | ||
+ | amounts was made and used to perform CFPS reactions. Two types of CFPS system were | ||
+ | used; one which contained cell extract from the same batch that was used to build the SRD | ||
+ | model (B1), and extract from a separate batch, but which was prepared in an identical way | ||
+ | (D1). The results showed that CFPS reactions using extract from the same batch that was | ||
+ | used to build the model (B1) did indeed increase the CFPS activity of that extract, and the | ||
+ | activity was within the confidence intervals (CIs) predicted by the model (Figure 3.3.3b). For | ||
+ | systems using a separate batch of extract (D1), the new supplement solution caused a | ||
+ | decrease in activity. This backs up previous research which suggests that | ||
+ | each batch of cell free extract requires its own optimal conditions for high protein synthesis | ||
+ | activity. It also shows that a multifactorial Design of Experiments approach can easily | ||
+ | determine important factors in CFPS systems, and accurately predict optimal supplement | ||
+ | amounts. | ||
+ | |||
+ | </p> | ||
Revision as of 11:21, 27 October 2017
Our Models |
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Multicellular Modelling: Simbiotics![]() Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. |
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Cell Free Protein Synthesis Systems Optimisation: Design of Experiments (JMP)![]() Diagrammatic Overview: This is a caption. This is a caption. This is a caption. This is a caption. This is a caption. This is a caption.
Previous research has shown that the concentration of some components of the supplement solution are crucial for efficient protein synthesis, and that for each batch of extract produced the optimal concentration may need to be found (Yang, et al., 2012). Studies which have explored this have only focused on, at most, a few components at a time (Garamella, et al., 2016; Kelwick, et al., 2016), which means that important interactions between the components may have been missed. In this study, a multifactorial approach will be taken to investigate the effect that all supplements have on the protein synthesis activity of CFPS systems simultaneously.
Traditionally, biologists tend to use One Factor At a Time (OFAT) approaches to determine
the effect and importance of factors on a system, which can sometimes be a poor method.
By only determining the effect that a single factor has on a system at a time, important
interactions can be missed. For example, removing only factor A may have no effect, and
removing only factor B may also have no effect, but removing both may cause an adverse
effect. Therefore, it is important to take a multifactorial approach when investigating the
importance of conditions or components of a system, or when trying to optimise a system.
An issue with this approach is that a large number of experiments may be required to fully
investigate all factors. By using statistical methods, a Design of Experiments (DoE) can be
determined which has the minimum number of experiments required to explore questions
such as the importance of factors in a system. This approach also allows for robustness
testing or determining batch-batch variation (Anderson & Whitcomb, 2010). As discussed
here [Link to Cell Free section of wiki], CFPS systems can be plagued with issues rising from
variation, so this approach offers a method to investigate the causes. It could also be used to
determine less important components of the supplement solution premix which is added to
CFPS systems, and hence a minimal supplement premix could be determined. Previous research has shown that the concentration of certain salts in the CFPS supplement premix are crucial for maximal protein synthesis activity [REF]. A Design of Experiments approach was used to determine which of the four salts (magnesium glutamate, potassium glutamate, sodium oxalate, and ammonium acetate) are the most important using the JMP software. A classical screening design was created with all four salts as continuous factors and CFPS activity as the response to be maximised. A concentration of ‘0’ was used as the lower limit for each factor, and the concentration used normally in CFPS supplement premixes was used as the upper limit (Figure 1). The screening design generated is shown in table 1. CFPS reactions were performed using supplement solution premixes with salt concentrations as determined by the main effects screening design. Reactions were incubated with 1.7 μg plasmid DNA encoding sfGFP (superfolder Green Fluorescent Protein) at 37 o C for 13 hours. CFPS activity was calculated as fluorescence intensity at 13 hours minus fluorescence intensity at 15 mins. This data was then used to generate a bar chart of Contrast values and a Half-Norma Plot (Figure 2 and 3) to determine which factors were having the most effect on CFPS activity. It should be noted that predictions for non-primary factors (i.e. interactions) may be inaccurate as they were forced-orthogonal. Considering the primary factors, magnesium glutamate was found to be the salt supplement with the largest contrast value, followed by potassium glutamate. This suggests that these two salt supplements were the most important. Sodium oxalate had a lower contrast value than either of the two glutamate salts, and was considered to have moderate importance in terms of CFPS activity. Ammonium acetate had an extremely low contrast value, suggesting that it may be unimportant for enhancing CFPS activity. The DoE software, JMP, was used to create a surface response design (SRD) for the three salts which were found by the screening design to have the most effect on CFPS activity (magnesium glutamate, potassium glutamate, and sodium oxalate). Ammonium acetate was kept at the default concentration and was not varied. Four SRDs were created using JMP; Central Composite Design-Uniform Precision design (CCD-UP), Box-Behnken (BB), Central Composite Design-Orthogonal (CCD-O), and Central Composite Design (CCD). The design diagnostics feature was used to compare the designs (Figure 4). Specifically, the colour map on correlations, power analysis for each factor and interaction, D, G, and A efficiencies, average variance of prediction, and number of reactions were compared to determine which design would be used. The colour map on correlations shows how correlated two terms are (red is highly correlated, blue is highly un-correlated). The more correlated two terms are, the more difficult it is to determine which is responsible for the effect on the response (Anderson & Whitcomb, 2010). As would be expected, in each design, terms are highly correlated with themselves (observed as a diagonal red line). Other terms are generally very lowly correlated with different terms. For the CCD-UP, BB, and CCD, the terms at the bottom right of the map have correlations above 0. For CCD-UP and BB, these correlations are still very low, but for CCD they are at about 0.5. Power analysis shows the likelihood of detecting an active effect for terms in the design (Anderson & Whitcomb, 2010). The CCD-O had a higher Power for all terms, with CCD-UP having the next highest. BB and CCD had lower Power for all terms, but some terms were higher in the BB design than the CC design, and some higher in CC design than the BB design.
D, G, and A efficiencies are a measure of each design to be D, G, and A optimised. A design
is D optimal if confidence regions for the vector of regression coefficients are minimized, G
optimal if maximum prediction variance over the design region is minimized, and A optimal if
the sum of the regression coefficient variance is minimized (Anderson & Whitcomb, 2010).
The CCD-UP design has the highest D efficiency and the BB design has the lowest. The
CCD design has the highest G efficiency and the BB design has the lowest. The CCD-UP
design has the highest A efficiency and the BB design has the lowest.
Results for the remaining reactions were used to build a model in JMP to predict an optimal
composition for the three salts. The model predicted that at high amounts, magnesium
glutamate and sodium oxalate were having an inhibitory effect, and potassium glutamate
was having an enhancing effect on CFPS activity (Figure 3.3.3a). It is well known that
magnesium ions are crucial for protein synthesis, for example in the functioning of
ribosomes, however at high amounts magnesium can become inhibiting to protein synthesis
by stalling translation at the translocation step (Li, et al., 2014). Therefore, it is not
unexpected that magnesium glutamate causes a decrease in protein synthesis activity at
certain concentrations. |