Team:HKU-HKBU/polar expression design

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=Polar Expression - Design=
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='''Design'''=
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Polar expression of a specific fragment of DNA in our project is crucial to achieve effective utilization of the propulsion force of the bacteria in the Bactomotor. Two polar expression systems were constructed here for various choices of strain selection.
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To achieve the interaction between bacteria and micromotors (bactomotor), polar expression of the protein streptavidin at one end of the bacteria is crucial in our project. When polar expression streptavidin bacteria encounter the biotin coated motor, there specific combination and bacteria mobility can generate propulsion force to the Bactomotor. Therefore, this synthetic device is capable to convert biological energy into mechanical work.  
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*In ''E.coli'' system
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Two polar expression systems were constructed as follows:
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*'''AIDA system'''
In the plasmid pET-SP-GFP-Streptavidin-AIDA, '''pET''' is a promoter which needs T7 polymerase to promote its function; '''Signal peptide'''(SP) can bring the plasmid to the membrane region of the bacteria; '''GFP''' is integrated within the plasmid for detection; '''Streptavidin''' is specifically binding with biotin, which achieved the combination of the bacteria and biotin coated motor; '''AIDA''' is a transmembrane protein, which leads to the polar expression of the whole plasmid. However, AIDA protein can only be expressed in a LPS complete strain, which is a constraint when choosing the bacteria strains.  
In the plasmid pET-SP-GFP-Streptavidin-AIDA, '''pET''' is a promoter which needs T7 polymerase to promote its function; '''Signal peptide'''(SP) can bring the plasmid to the membrane region of the bacteria; '''GFP''' is integrated within the plasmid for detection; '''Streptavidin''' is specifically binding with biotin, which achieved the combination of the bacteria and biotin coated motor; '''AIDA''' is a transmembrane protein, which leads to the polar expression of the whole plasmid. However, AIDA protein can only be expressed in a LPS complete strain, which is a constraint when choosing the bacteria strains.  
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*In ''Salmonella'' system
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*'''Lpp-OmpA system'''
The plasmid we used is Lpp-OmpA-GFP-streptavidin, which can be polarly expressed on one side of rod-shaped bacteria. '''Lpp''' functions as a signal peptide; '''OmpA''' is the player which can achieve the surface expression of specific proteins; '''GFP-Strp''' acts the same role as above.  
The plasmid we used is Lpp-OmpA-GFP-streptavidin, which can be polarly expressed on one side of rod-shaped bacteria. '''Lpp''' functions as a signal peptide; '''OmpA''' is the player which can achieve the surface expression of specific proteins; '''GFP-Strp''' acts the same role as above.  
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After we successfully constructed two polar expression systems, strain selection became a
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After we successfully constructed two polar expression systems, which bacteria strain to be used becomes our crutial task.
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=Strain Selection=
=Strain Selection=
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To maximize the efficiency of motor, it is essential to screen and select bacteria species and strains equipped with outstanding swimming abilities as well as a complete LPS (lipopolysaccharide) layer. This layer, present in the bacterial cell wall, is required for polar expression of certain proteins(AIDI) The candidates included ''Escherichia. Coli'' strains: ''BL21, NCM3722, MG1655, MG3,'' and ''2443'' (strain 2443 ompT pIB264), and ''Salmonella SL7207''.
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*'''For AIDA system'''  
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===Swimming plate assay===
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The fastest-swimming bacteria among our candidates were determined by this assay. By measuring the diameter on the swimming plate at differnt Different strains were first introduced to suitable agar media, and the diameter of the colonies are measured every hour in the first 8 hours in order to obtain approximated data of their swimming speed. The data are also recorded overnight for confirmation. No or inconspicuous increase in diameter is classified as negative result. The swim plates of BL21, NCM3722 and MG1655 yielded negative results. Although the clouded area of the MG3 plate showed augmentation in diameter, the colony was of the shape of dispersed clouds whereas a positive result would show distinct  circles as a result of chemotaxis. We attribute this to contamination by other bacteria. Salmonella - a bacteria with well known swimming abilities (~4.5mm/hr) - was employed as our positive control. However, surprisingly, E.coli 2443 shows 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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{| align="center" border="1px" cellpadding="4px"
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! Strain
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| BL21 || NCM3722 || MG1655 || MG3 || 2443 || SL7207
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!Swim plate assay result
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| - || - || - || / || ++ || +
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===LPS completeness===
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LPS takes vital part in specifically expressing streptavidin, the protein enabling the binding of bacteria to motor in our project. After literature reviews, ''E. coli 2443'' [[#Reference | [1]]] and ''Salmonella'' [[#Reference | [2]]] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.
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===Conclusion===
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Combining the results of two parts, ''E. coli 2443'' and ''Salmonella SL7207'' are adopted in further experiment and entitled with PB (Propeller Bacteria) 001 and PB 002 correspondingly.
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==Reference==
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To achieve polar expression, it is essential to screen and select bacteria species and strains equipped with outstanding swimming abilities as well as a complete LPS (lipopolysaccharide) layer. This layer, present in the bacterial cell wall, is required for polar expression of certain proteins(AIDI) The candidates included ''Escherichia. Coli'' strains: ''BL21, NCM3722, MG1655, MG3,'' and ''2443'' (strain 2443 ompT pIB264), and ''Salmonella SL7207''.
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# 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
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# 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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*'''For Lpp-OmpA system'''
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Lpp-OmpA system can only achieve outer membrane surface expression. To select the strain which can polar express this plasmid, Lpp-OmpA were transformed into the strains above. GFP were fused into this plasmid for the conviniece of detection under fluorescent microscope.
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{{Team:HKU-HKBU/footer}}
 
   
   

Latest revision as of 15:25, 14 October 2009

Design

To achieve the interaction between bacteria and micromotors (bactomotor), polar expression of the protein streptavidin at one end of the bacteria is crucial in our project. When polar expression streptavidin bacteria encounter the biotin coated motor, there specific combination and bacteria mobility can generate propulsion force to the Bactomotor. Therefore, this synthetic device is capable to convert biological energy into mechanical work.

Two polar expression systems were constructed as follows:

  • AIDA system

In the plasmid pET-SP-GFP-Streptavidin-AIDA, pET is a promoter which needs T7 polymerase to promote its function; Signal peptide(SP) can bring the plasmid to the membrane region of the bacteria; GFP is integrated within the plasmid for detection; Streptavidin is specifically binding with biotin, which achieved the combination of the bacteria and biotin coated motor; AIDA is a transmembrane protein, which leads to the polar expression of the whole plasmid. However, AIDA protein can only be expressed in a LPS complete strain, which is a constraint when choosing the bacteria strains.

  • Lpp-OmpA system

The plasmid we used is Lpp-OmpA-GFP-streptavidin, which can be polarly expressed on one side of rod-shaped bacteria. Lpp functions as a signal peptide; OmpA is the player which can achieve the surface expression of specific proteins; GFP-Strp acts the same role as above.

After we successfully constructed two polar expression systems, which bacteria strain to be used becomes our crutial task.

Strain Selection

  • For AIDA system

To achieve polar expression, it is essential to screen and select bacteria species and strains equipped with outstanding swimming abilities as well as a complete LPS (lipopolysaccharide) layer. This layer, present in the bacterial cell wall, is required for polar expression of certain proteins(AIDI) The candidates included Escherichia. Coli strains: BL21, NCM3722, MG1655, MG3, and 2443 (strain 2443 ompT pIB264), and Salmonella SL7207.

  • For Lpp-OmpA system

Lpp-OmpA system can only achieve outer membrane surface expression. To select the strain which can polar express this plasmid, Lpp-OmpA were transformed into the strains above. GFP were fused into this plasmid for the conviniece of detection under fluorescent microscope.



HKU-HKBU polar expression design.png

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