Difference between revisions of "Part:BBa K4115017"
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<p align="justify">
 
<p align="justify">
 
<b>Methods/Protocols</b><br>
 
<b>Methods/Protocols</b><br>
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1. Before culturing, E.coli is stored at 4 degrees. Culture experiment group(pUC-SacC) and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps. <br>
+
1. Before culturing, E.coli is stored at 4 degrees. Culture experiment group(pUC-SacC, The plasmids carried by the engineering bacteria in this experiment have been registered, numbered <partinfo>BBa_K4115036</partinfo>) and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps. <br>
 
2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L.<br>
 
2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L.<br>
 
3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 80ul liquids for tests.  
 
3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 80ul liquids for tests.  
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The experimental group(pUC-SacC, shown by the blue line on the figure) grows faster at the beginning(8~18h). After that, the experimental group reached the stationary phase at OD600~2.0, while the control group was still growing slowly until OD600~3.0.  
 
The experimental group(pUC-SacC, shown by the blue line on the figure) grows faster at the beginning(8~18h). After that, the experimental group reached the stationary phase at OD600~2.0, while the control group was still growing slowly until OD600~3.0.  
 
In conclusion, the expression of SacC accelerates the growth of E.coli, but makes E.coli reach the stationary phase at a lower OD600.<br>
 
In conclusion, the expression of SacC accelerates the growth of E.coli, but makes E.coli reach the stationary phase at a lower OD600.<br>
−
We are confused about the differences in the stationary phase. One hypothesis is that the expression of SacC causes some metabolism burden. Considering J23101 is a relatively strong constitutive promoter in the Anderson library, I decided to construct two more plasmids, using J23107 and J23109 to express SacC. The plasmids construction process is similar to that of pUC-Empty and pUC-SacC(full length).
+
We are confused about the differences in the stationary phase. One hypothesis is that the expression of SacC causes some metabolism burden. Considering J23101 used in <partinfo>BBa_K4115036</partinfo>is a relatively strong constitutive promoter in the Anderson library, I decided to construct two more plasmids, using J23107 and J23109 to express SacC. The plasmids construction process is similar to that of pUC-Empty and pUC-SacC(full length).
 
</p>
 
</p>
 +
===Replacements of J23101===
 +
<p>
 +
Since constitutive promoters in Anderson library only have several base pairs differences from each other, we directly use PCR to introduce the site-specific mutations to the promoter region.
 +
</p>
 +
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4115017 parameters</partinfo>
 
<partinfo>BBa_K4115017 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 07:44, 3 October 2022


SacC (the extracellular sucrase gene)

The sequence of Zymomonas mobilis gene sacC that encodes the extracellular sucrase. Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1309
    Illegal PstI site found at 437
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1352
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 437
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

The expression product is exhibited sucrase but not levansucrase activity,which means it behaves like a true sucrase. Heterologous expression of SacC aims to improve the utilization of sucrose in the medium by E. coli responsible for the synthetic production of biologics.

Cultivation, Experiment and Improvement Process

100 mL Fermentation E. coli DH5α with BBa_K4115017

Figure 1: Fermentation of E. coli with BBa_K4115017 in 300ml conical flask in biological shaker, scale: 100 mL M9 medium (with 20% sucrose), 37 °C, 220 rpm, initial pH 7.5, OD600 measured every 2 hours.

Methods/Protocols
1. Before culturing, E.coli is stored at 4 degrees. Culture experiment group(pUC-SacC, The plasmids carried by the engineering bacteria in this experiment have been registered, numbered BBa_K4115036) and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps.
2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L.
3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 80ul liquids for tests.






Analysis

The experimental group(pUC-SacC, shown by the blue line on the figure) grows faster at the beginning(8~18h). After that, the experimental group reached the stationary phase at OD600~2.0, while the control group was still growing slowly until OD600~3.0. In conclusion, the expression of SacC accelerates the growth of E.coli, but makes E.coli reach the stationary phase at a lower OD600.
We are confused about the differences in the stationary phase. One hypothesis is that the expression of SacC causes some metabolism burden. Considering J23101 used in BBa_K4115036is a relatively strong constitutive promoter in the Anderson library, I decided to construct two more plasmids, using J23107 and J23109 to express SacC. The plasmids construction process is similar to that of pUC-Empty and pUC-SacC(full length).

Replacements of J23101

Since constitutive promoters in Anderson library only have several base pairs differences from each other, we directly use PCR to introduce the site-specific mutations to the promoter region.