Latest revision as of 16:41, 12 October 2009

Strain Selection

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. Our preliminary candidates included Escherichia. Coli strains: BL21, NCM3722, MG1655, MG3, and 2443 (strain 2443 ompT pIB264), and Salmonella SL7207.

Swim plate assay

This determines the fastest-swimming bacteria among our candidates. Different strains are 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.

Strain	BL21	NCM3722	MG1655	MG3	2443	SL7207
Swim plate assay result	-	-	-	/	++	+

LPS completeness

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 [1] and Salmonella [2] are identified to possess complete LPS layer. Their ability to express desirable proteins on the head is examined in later experiment.

Conclusion

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.

Reference

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
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

@@ Line 4: / Line 4: @@
 =Strain Selection=
-To maximize the efficiency of motor, it is pivotal to screen and select bacteria species and strains that are equipped outstanding swimming ability as well as complete LPS – lipopolysaccharide, a layer in bacterial cell wall required for polar expression of certain proteins. Our preliminary candidates include ''Escherichia. Coli'' strains: BL21, NCM3722, MG1655, MG3, and 2443 (strain 2443 ompT pIB264), and ''Salmonella'' SL7207.
+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. Our preliminary candidates included ''Escherichia. Coli'' strains: BL21, NCM3722, MG1655, MG3, and 2443 (strain 2443 ompT pIB264), and ''Salmonella'' SL7207.
 ===Swim plate assay===
-Swim plate assay is applied to select the best swimmer among our candidates. Their colonies diameter is measured every hour in the first 8 hours, for attaining approximate data of their swimming speed, and after overnight to reconfirm. No or inconspicuous increase in diameter is classified as negative result; those that exhibit negative swim ability consist of BL21, NCM3722, and MG1655. Although MG3 shows augmentation in diameter, its colony shapes as dispersed cloud instead of disk with distinct chemotaxis circles. This phenomenon possibly stems from contamination of undesirable bacteria. Swim ability of Salmonella is widely known, due to its function of positive control employed in swim plate assay. Moreover, surprisingly, E.coli 2443 manifests even more impressive performance, with a speed of approximate 5.5mm increase in radius every hour at the end of eight-hour-experiment (for comparison, the speed of Salmonella is 4.5mm/hr).
+This determines the fastest-swimming bacteria among our candidates. Different strains are 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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Team:HKU-HKBU/strain selection

From 2009.igem.org