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Team:Washington/Alkanes/Future/Vector
From 2011.igem.org
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==Use of Different strains== | ==Use of Different strains== | ||
We had suspected that different strains of ''E. coli'' would produce varying amounts of alkane. Initial experiments were done in MG1655, and we decided to test XL-1 blue( a commercial supercopentent variant of DH5a) for the ability to produce alkane. | We had suspected that different strains of ''E. coli'' would produce varying amounts of alkane. Initial experiments were done in MG1655, and we decided to test XL-1 blue( a commercial supercopentent variant of DH5a) for the ability to produce alkane. | ||
| - | [[File:Washinton_XL21vsMG1655.png|center|500px|thumb| | + | [[File:Washinton_XL21vsMG1655.png|center|500px|thumb|XL-1 blue produces more alkane than MG1655]] |
XL-1 blue was able to produce more alkane than MG1655. In addition, XL-1 blue did not produce detectable levels of C16 alcohol. This makes C17 alkene analysis easier, and would mean a purer alkane product. | XL-1 blue was able to produce more alkane than MG1655. In addition, XL-1 blue did not produce detectable levels of C16 alcohol. This makes C17 alkene analysis easier, and would mean a purer alkane product. | ||
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==Sealed vs. Open Tubes== | ==Sealed vs. Open Tubes== | ||
We were concerned that alkane evaporation may have been evaporating, reducing apparent yield. Therefore, we performed tests were the tubes were either capped like normal, or capped with foil coverting the opening, redcing evaporation. | We were concerned that alkane evaporation may have been evaporating, reducing apparent yield. Therefore, we performed tests were the tubes were either capped like normal, or capped with foil coverting the opening, redcing evaporation. | ||
| - | [[File:Washinton_open_sealed.png|center|500px|thumb| | + | [[File:Washinton_open_sealed.png|center|500px|thumb|Sealing culture tubes increases yield.]] |
Covered tubes showed significantly more alkane, so all further tests would be conducted in sealed tubes. | Covered tubes showed significantly more alkane, so all further tests would be conducted in sealed tubes. | ||
==Aerobic vs Microaerobic growth== | ==Aerobic vs Microaerobic growth== | ||
In many industrial production applications, growth in conditions with little or no oxygen can improve yield. Therefore, we tested alkane yield in cultures grown in airtight vials with only a small amount of air on the top in order to severely limit oxygen availability. | In many industrial production applications, growth in conditions with little or no oxygen can improve yield. Therefore, we tested alkane yield in cultures grown in airtight vials with only a small amount of air on the top in order to severely limit oxygen availability. | ||
| - | [[File:Washington_aerobicVsMicroaerobic.png|center|500px|thumb| | + | [[File:Washington_aerobicVsMicroaerobic.png|center|500px|thumb|Oxygen has a positive effect on alkane production]] |
Based upon this data, aerobic growth appears to be better for alkane yield. Note that this difference is not likely due to differences in growth rate, as both cultures were inoculated to a high OD600 of 1. | Based upon this data, aerobic growth appears to be better for alkane yield. Note that this difference is not likely due to differences in growth rate, as both cultures were inoculated to a high OD600 of 1. | ||
==pSB1C3-ADC-AAR vs pSB3k3-ADC/pSB4C5-AAR== | ==pSB1C3-ADC-AAR vs pSB3k3-ADC/pSB4C5-AAR== | ||
The original study expressed ADC and AAR on seperate low copy number vectors. If we could get high alkane yields using low copy number vectors, the lesser protien and vector production would make the use of a lower copy number vector preferable. | The original study expressed ADC and AAR on seperate low copy number vectors. If we could get high alkane yields using low copy number vectors, the lesser protien and vector production would make the use of a lower copy number vector preferable. | ||
| - | + | [[File:Washington_copynumber.png|center|500px|thumb|greater ADC/AAR expression results in more alkane production.]] | |
| - | + | The fact that high copynumber vector use results in an increase in yield means that high expression levels of ADC and/or AAR are required for high alkane yields. | |
TB vs M9 | TB vs M9 | ||
Revision as of 23:32, 18 September 2011
Growth Optimization
Using our initial, non-optimized growth conditions, we were able to obtain alkane/ene yields of approximately 2 mg/L. In order to make analysis easier, and to make it easier to determine the effects of the addition of additional modules, we wanted to increase yield. Our efforts focused on how by varying system conditions, we could increase yield.
Effect of Trace metals on yield
We had suspected that the trace metals in the original media may be inhibiting alkane production. We analyzed alkane production of cells transformed with the basic Petrobrick in medias of varying trace metal concentration.
Based upon this test, we determined that trace metals have a negative effect on alkane yield, and that all future tests would be conducted in an M9 media without additional trace metals.
Use of Different strains
We had suspected that different strains of E. coli would produce varying amounts of alkane. Initial experiments were done in MG1655, and we decided to test XL-1 blue( a commercial supercopentent variant of DH5a) for the ability to produce alkane.
XL-1 blue was able to produce more alkane than MG1655. In addition, XL-1 blue did not produce detectable levels of C16 alcohol. This makes C17 alkene analysis easier, and would mean a purer alkane product.
Sealed vs. Open Tubes
We were concerned that alkane evaporation may have been evaporating, reducing apparent yield. Therefore, we performed tests were the tubes were either capped like normal, or capped with foil coverting the opening, redcing evaporation.
Covered tubes showed significantly more alkane, so all further tests would be conducted in sealed tubes.
Aerobic vs Microaerobic growth
In many industrial production applications, growth in conditions with little or no oxygen can improve yield. Therefore, we tested alkane yield in cultures grown in airtight vials with only a small amount of air on the top in order to severely limit oxygen availability.
Based upon this data, aerobic growth appears to be better for alkane yield. Note that this difference is not likely due to differences in growth rate, as both cultures were inoculated to a high OD600 of 1.
pSB1C3-ADC-AAR vs pSB3k3-ADC/pSB4C5-AAR
The original study expressed ADC and AAR on seperate low copy number vectors. If we could get high alkane yields using low copy number vectors, the lesser protien and vector production would make the use of a lower copy number vector preferable.
The fact that high copynumber vector use results in an increase in yield means that high expression levels of ADC and/or AAR are required for high alkane yields. TB vs M9
and more...
