Team:HKU-HKBU/Polar Expression Results

From 2009.igem.org

(Difference between revisions)
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===Swimming Ability Assay===
===Swimming Ability Assay===
-
The purpose of this part is to find one or servel strains of bacteria which could swim. Therefore, we tested bacteria of ''E.coli'' and ''Salmonella''. The swimming plates of ''BL21'', ''NCM3722'' and ''MG3'' yielded negative results. We found ''Salmonella'' - we named it ''YBS01'' is a bacteria with high swimming abilities (~4.5mm/hr), and a kind of ''E.coli 2443'', we named it ''YBE01'' showed even more impressive performance, with a speed of approximate 5.5mm increase in radius every hour at the end of eight-hour-experiment.
+
The purpose of this part is to find one or several strains of bacteria which could swim. Therefore, we tested bacteria of ''E.coli'' and ''Salmonella''. The swimming plates of ''BL21'', ''NCM3722'' and ''MG3'' yielded negative results. We found a ''Salmonella'' strain - we named it ''YBS01'' is a bacteria with high swimming abilities (~4.5mm/hr), and a kind of ''E.coli 2443'', we named it ''YBE01'' showed even more impressive performance, with a speed of approximate 5.5mm increase in radius every hour at the end of eight-hour-experiment.
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===LPS Completeness Search===
===LPS Completeness Search===
-
LPS takes vital part in AIDA polar expression system. Due to many mutations exist in different strains of ''E.coli'' or ''Salmonella'', some of these mutations may cause the defcient in there LPS layers. There mutants could survival in the culture. But for AIDA expression, when the host's LPS is incomplete, the AIDA will express all over the bacteria surface; when the host's LPS is complete, the AIDA will express only on one side of the bacteria. After literature reviews, ''E. coli 2443''-''YBE01'' [[#Reference | [1]]] and ''Salmonella''-''YBS01'' [[#Reference | [2]]] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.
+
LPS takes vital part in AIDA polar expression system. Due to many mutations exist in different strains of ''E.coli'' or ''Salmonella'', some of these mutations may cause the defciency in there LPS layers. There mutants could survive in the culture. But for AIDA expression, when the host's LPS is incomplete, the AIDA will express all over the bacteria surface; when the host's LPS is complete, the AIDA will express only on one side of the bacteria. After literature reviews, ''E. coli 2443''-''YBE01'' [[#Reference | [1]]] and ''Salmonella''-''YBS01'' [[#Reference | [2]]] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.
='''Polar Expression'''=
='''Polar Expression'''=
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===Fluorescent Microscope===
===Fluorescent Microscope===
-
After literature review, we found the ''E.coli BL21'' was also LPS complete strains. Therefore, we used the plasmid containing T7 promoter which controlled the expression of '''GFP-Strp-AIDA'''. ''BL21'' strain contained T7 polymerase, therefore when the IPTG was added into the culture medium, '''GFP-Strp-AIDA''' would be strongly induced. In the figure below, it showed the expression of '''GFP'''. This expression under the fluorescent microscope using oil lens with the manification of 600 times. In the merge picture of dark field and FITC field, the fluorescent proteins were showed at one end of the bacteria.
+
After literature review, we found the ''E.coli BL21'' was also LPS complete strains. Therefore, we used the plasmid containing T7 promoter which controlled the expression of '''GFP-Strp-AIDA'''. ''BL21'' strain contained T7 polymerase, therefore when the IPTG was added into the culture medium, '''GFP-Strp-AIDA''' would be strongly induced. In the figure below, it showed the expression of '''GFP'''. This expression was observed under the fluorescent microscope using oil lens with the manification of 600 times. In the merge picture of dark field and FITC field, the fluorescent proteins were showed at one end of the bacteria.
Line 32: Line 32:
-
For this project of bactomotor, we required the one side expression of specific proteins and the swimming ability of the bacteria at the same time. However, the ''BL21'' strain couldn't swim. Therefore we need to apply this system into our new candidate ''YBE01''. While ''YBE01'' didn't contain T7 polymerase, being not able to activate T7 promoter in this plasmid. Therefore, a new plasmid with strongly inducible or consitutive expression system should be constructed. Attemps were made to use '''pcI857''' was thermo-inducible promoter which was turn-on at 37 centigrade degree and turn-off at 30 centigrade degree. We also tried '''plac''' without '''lacI''' as a contitutive promoter. However, only weak and unstable expression of '''GFP''' could be observed of these systems. One of the possible expalanation could be that these two systems were not strong or stable enough to ensure the consistant expression of our required proteins.
+
For this project, we required a strain that expresses specific proteins in only one side, and has swimming ability at the same time. However, the ''BL21'' strain couldn't swim. Therefore we need to apply one side expression system into our new candidate ''YBE01''. While ''YBE01'' didn't contain T7 polymerase, being not able to activate T7 promoter in this plasmid. Therefore, a new plasmid with strongly inducible or constitutive expression system should be constructed. Attempts were made to use '''pcI857''' as thermo-inducible promoter which was turn-on at 37 celsius degree and turn-off at 30 celsius degree. We also tried '''plac''' without '''lacI''' as a constitutive promoter. However, only weak and unstable expression of '''GFP''' could be observed. One of the possible explanations could be that these two systems were not strong or stable enough to ensure the consistent expression of our required proteins.
===SDS PAGE and Western Blotting===
===SDS PAGE and Western Blotting===
-
The surface expression of streptavidin was essential to specificly binding with biotin-coated motor. To prove that the '''GFP-streptavidin''' was expressed on the outer membrane of the bacteria through''' AIDA''' system, different contents of the bacteria should be separated by ultracentrifuge. After separation, the outer membrane, inner membrane, total membrane and cytoplasm were collected as different samples. SDS PAGE and Western Blotting were conducted for verification. Each sample was quantified by BCA assay to make sure the equal loading of proteins. The western blotting results showed most '''GFP''' proteins were localized on the outer membrane of the bacteria, which corresponded with the expected result that the specific expression of proteins were on the outer membrane.
+
The surface expression of streptavidin was essential to specifically bind to biotin-coated motor. To prove that the '''GFP-streptavidin''' was expressed on the outer membrane of the bacteria through''' AIDA''' system, different contents of the bacteria were separated by ultracentrifuge. After separation, the outer membrane, inner membrane, total membrane and cytoplasm were collected as different samples. SDS PAGE and Western Blotting were conducted for verification. Each sample was quantified by BCA assay to make sure the equal loading of proteins. The western blotting results showed most '''GFP''' proteins were localized on the outer membrane of the bacteria, which corresponded with the expected result that the specific expression of proteins were on the outer membrane.

Revision as of 12:03, 21 October 2009

Contents

Strain Selecion

Swimming Ability Assay

The purpose of this part is to find one or several strains of bacteria which could swim. Therefore, we tested bacteria of E.coli and Salmonella. The swimming plates of BL21, NCM3722 and MG3 yielded negative results. We found a Salmonella strain - we named it YBS01 is a bacteria with high swimming abilities (~4.5mm/hr), and a kind of E.coli 2443, we named it YBE01 showed even more impressive performance, with a speed of approximate 5.5mm increase in radius every hour at the end of eight-hour-experiment.


HKU-BU-Swimming assay second.png


LPS Completeness Search

LPS takes vital part in AIDA polar expression system. Due to many mutations exist in different strains of E.coli or Salmonella, some of these mutations may cause the defciency in there LPS layers. There mutants could survive in the culture. But for AIDA expression, when the host's LPS is incomplete, the AIDA will express all over the bacteria surface; when the host's LPS is complete, the AIDA will express only on one side of the bacteria. After literature reviews, E. coli 2443-YBE01 [1] and Salmonella-YBS01 [2] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.

Polar Expression

AIDA Polar Expression System

Fluorescent Microscope

After literature review, we found the E.coli BL21 was also LPS complete strains. Therefore, we used the plasmid containing T7 promoter which controlled the expression of GFP-Strp-AIDA. BL21 strain contained T7 polymerase, therefore when the IPTG was added into the culture medium, GFP-Strp-AIDA would be strongly induced. In the figure below, it showed the expression of GFP. This expression was observed under the fluorescent microscope using oil lens with the manification of 600 times. In the merge picture of dark field and FITC field, the fluorescent proteins were showed at one end of the bacteria.


HKU-HKBU polar expression results E coli fluorescent3.png


For this project, we required a strain that expresses specific proteins in only one side, and has swimming ability at the same time. However, the BL21 strain couldn't swim. Therefore we need to apply one side expression system into our new candidate YBE01. While YBE01 didn't contain T7 polymerase, being not able to activate T7 promoter in this plasmid. Therefore, a new plasmid with strongly inducible or constitutive expression system should be constructed. Attempts were made to use pcI857 as thermo-inducible promoter which was turn-on at 37 celsius degree and turn-off at 30 celsius degree. We also tried plac without lacI as a constitutive promoter. However, only weak and unstable expression of GFP could be observed. One of the possible explanations could be that these two systems were not strong or stable enough to ensure the consistent expression of our required proteins.

SDS PAGE and Western Blotting

The surface expression of streptavidin was essential to specifically bind to biotin-coated motor. To prove that the GFP-streptavidin was expressed on the outer membrane of the bacteria through AIDA system, different contents of the bacteria were separated by ultracentrifuge. After separation, the outer membrane, inner membrane, total membrane and cytoplasm were collected as different samples. SDS PAGE and Western Blotting were conducted for verification. Each sample was quantified by BCA assay to make sure the equal loading of proteins. The western blotting results showed most GFP proteins were localized on the outer membrane of the bacteria, which corresponded with the expected result that the specific expression of proteins were on the outer membrane.


HKU-BU-AIDA-west2.png


When observing the moving strains in the microscope, we could find that all the GFP were expressed at the forehead end of the rod shaped bacteria. Therefore, this system was a perfect choice for binding with biotin-coated motor. It can express streptavidin at pole(s), which allows cells to adhere in the same orientation to a microrotatory motor through biotin-streptavidin interaction.

Lpp-OmpA System

Fluorescent Microscope

For AIDA system, the requirement for strains were critical, such as LPS completeness, strong activators, etc. Another polar expression system were constructed for back-up plan. Lpp consists of 9 amino acids of signal peptide for targeting the inner membrane of the bacteria. OmpA (91-261) is a 5-cross trans-membrane proteins which could bring cargos to the outer membrane of the bacteria. We constructed a plasmid by fusing GFP-strp with Lpp-OmpA system.

The surface expression of Lpp-ompA-GFP-Strp were consistent, however the polar expression of this plasmid couldn't be ensured. Therefore, this plasmid was transformed into various strains of E.coli and Salmonella. We found that in the stage phase of YBS01, polar expression of the plamid was observed.

The left panel showed the polar expression of the plasmid in the stationary phase of YBS01 as a negative control. The right panel showed the merge picure of the bright field and FITC field under the fluorescnet microscopde with a manification of 600 times. The result indicated that polar expression of proteins were achieved by this system.


HKU-BU-lpp5.png


Western Blotting

To verify this hypothesis in a more convincing way, Western Blotting was conducted, which could reflect the surface expression of proteins. By studying the relative concentrations of expressed protein GFP in different sampls, a obvious darker band was observed in the outer membrane samples. This results corresponded with the hypothesis that the GFP proteins are particularly expressed on the outer membrane.


HKU-HKBU polar expression results Salmonella western3.png


Reference

  1. Sumita Jain, Peter van Ulsen, Inga Benz, M. Alexander Schmidt, Rachel Fernandez, Jan Tommassen, and Marcia B. Goldberg, Polar Localization of the Autotransporter Family of Large Bacterial Virulence Proteins, Journal of Bacteriology, July 2006, p. 4841-4850, Vol. 188, No. 13
  2. Maurien M. A. Olsthoorn, Bent O. Petersen, Siegfried Schlecht, Johan Haverkamp, Klaus Bock, Jane E. Thomas-Oates and Otto Holst, Identification of a Novel Core Type in Salmonella Lipopolysaccharide, The Journal of Biological Chemistry, Vol. 273, No. 7, Issue of February 13, pp. 3817-3829, 1998

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