Difference between revisions of "Part:BBa K3552000"
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We first constructed the GsPilA by SOE PCR through 8 different primers to maintain the exactly same amino acid sequence as in the Geobacter sulfurreducens. After the final construction of the generator of PilA, we added six His-tag to the GsPilA and expressed the gene in vivo in E.coli BL21. After 48 hours of growth, we extracted the pili and verified the existence of GsPilA on the outer cell through Western Blot. We also do electronic speculum and see the clear image of the pili in the solution.
 
We first constructed the GsPilA by SOE PCR through 8 different primers to maintain the exactly same amino acid sequence as in the Geobacter sulfurreducens. After the final construction of the generator of PilA, we added six His-tag to the GsPilA and expressed the gene in vivo in E.coli BL21. After 48 hours of growth, we extracted the pili and verified the existence of GsPilA on the outer cell through Western Blot. We also do electronic speculum and see the clear image of the pili in the solution.
  
−
To get a complete pili production system, several steps were included in the construction. We first ordered oligo DNAs from Gs pilA gene and assembled them by SOE PCR to acquired its completely same amino acid sequence as expressed by EHEC genes. Then we constructed the generator of the pili and obtain a complete circuit of <partinfo>BBa_K3552010</partinfo> .
+
To get a complete pili production system, we first ordered oligo DNAs from Gs pilA gene and assembled them by SOE PCR to acquired its completely same amino acid sequence as expressed by EHEC genes. Then we constructed the generator of the pili and obtain a complete circuit of <partinfo>BBa_K3552010</partinfo> .
  
−
For pili expression, there are several options of E.coli strains: BW25113, BL21, and PAP105. We chose E.coli BL21 for our pili expression because it is the best fit to our project as it has the highest pili yield among them and is easy to obtain. On the cultivation part, we cultured the bacteria in solid M9 medium because M9 has a clean content background and is relatively infertile, and the bacteria can be able to attach on the surface of the solid medium, which can stimulate the pili growth on bacteria membrane. Additionally, we provide the bacteria with glycerol as the carbon source because it was confirmed to help improve the conductivity of the pili produced.
+
For pili expression, we chose E.coli BL21 because it is the best fit to our project as it has the highest pili yield among them and is easy to obtain. On the cultivation part, we cultured the bacteria in solid M9 medium. Additionally, we provide the bacteria with glycerol as the carbon source because it was confirmed to help improve the conductivity of the pili produced.
  
 
We transformed <partinfo>BBa_K3552010</partinfo>  into E.coli BL21. The bacteria was first cultivated on a LB medium plate with kanamycin at 30°C for 24h. Then, we scraped the E.coli off the LB plates with 6ml liquid M9 solution and then coated the collected bacteria solution on 20 M9 medium plates with IPTG and kanamycin. Finally, the 20 coated M9 plates were cultured at 30°C for 48h.
 
We transformed <partinfo>BBa_K3552010</partinfo>  into E.coli BL21. The bacteria was first cultivated on a LB medium plate with kanamycin at 30°C for 24h. Then, we scraped the E.coli off the LB plates with 6ml liquid M9 solution and then coated the collected bacteria solution on 20 M9 medium plates with IPTG and kanamycin. Finally, the 20 coated M9 plates were cultured at 30°C for 48h.
Line 54: Line 54:
 
We harvested the bacteria from M9 plates after 48h cultivation by scrapping them off again with liquid M9 solution and collect the bacteria solution. By conducting the extraction and purification on the samples, we managed to get an amount Gs pili solution dissolving in 150mM ethanol-amine after filtration with a 100kDa membrane in nitrogen gas.  
 
We harvested the bacteria from M9 plates after 48h cultivation by scrapping them off again with liquid M9 solution and collect the bacteria solution. By conducting the extraction and purification on the samples, we managed to get an amount Gs pili solution dissolving in 150mM ethanol-amine after filtration with a 100kDa membrane in nitrogen gas.  
  
−
For physical confirmation of e-pili produced, we ordered a SEM Photography for our pili On the photographs, we can see that the pili was distributed evenly on the surface in porous structures between each pili individuals. The pili seemed to have a structure differ from the common structure of protein. This porous structure of the pili membrane is like sponge that can assure a water potential gradient would form within the pili membrane, which is crucial and beneficial for e-pili to generate electricity.
+
We also ordered a SEM Photography for our pili on the photographs, we can see that the pili was distributed evenly on the surface in porous structures between each pili individuals. In order to confirm the E.coli produced the correct e-pili, we add 6xHis-tags to the end of the Gs pilA gene sequence, and constructed a new plasmid. And the addition of His-tags will not affect the conductivity and electricity generation of the e-pili produced. Then, we conducted Western Blot experiment to convince the production. According to the positive strands on the membrane, Pili was confirmed to be produced and purified successfully, with an expected molecular mass of 10kDa.
−
It is hard to affirm that the pili was presented in the product we harvested without any evidence. In order to confirm the E.coli produced the correct e-pili, we ameliorate the sub-plasmid pilA-hofB-hofC by adding 6xHis-tags to the end of the Gs pilA gene sequence, and constructed a new major plasmid. And the addition of His-tags will not affect the conductivity and electricity generation of the e-pili produced. Then, we conducted Western Blot experiment, using the his-tag antibody attaching the his-tags on pili for coloration. According to the positive strands on the membrane, Pilin was confirmed to be produced and purified successfully, with an expected molecular mass of 10kDa.
+
We also conducted experiments to measure the voltages of each batteries. First we manufactured six standard electrodes by using three pili, two pieces each, all with triple layer of pili covered. Then we measured the voltage and compared the result which shows that Gm pili has the highest value of measured voltage where Gs pili has the least.
−
To further discover the spatial structure of the e-pili, we ordered a TEM Photography as well for our purified pili. But unfortunately, we haven’t explored a way of presenting the pili by TEM.
+
 
 +
In order to compare the pili yield, we established a new measurement for a quicker, clearer, and more accurate determination of pili production. We use his-tags antibodies to attach on the his-tags on the pili, and then the secondary antibodies will attached to the his-tags antibodies for coloration directly on the outer membrane of the bacteria. We analyse the datas and included the effects of two variables on pili production: cultivation time and mobile oxygen presence. The result shows a ranking from Gs pili to Pa pili to Gm pili.
  
 
<partinfo>BBa_K3552000 parameters</partinfo>
 
<partinfo>BBa_K3552000 parameters</partinfo>
 
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Revision as of 04:49, 26 October 2020


GsPilA

GsPilA is a type 4 pilus generally on Geobacter sulfurreducens which can conduct electricity. This part is in the part collection where we provide different conductive pilus based on the same pilin generator. By these different type 4 pilus we can compare the qualities.

The part collection includes: Parts that are different kinds of type 4 pilus: BBa_K3552000 BBa_K3552001 BBa_K3552002. Parts that are the generator of the type 4 pilus: BBa_K3552003 BBa_K3552004 BBa_K3552005 BBa_K3552006 BBa_K3552007 BBa_K3552008 BBa_K3552018 BBa_K3552019 BBa_K3552020 BBa_K3552021 BBa_K3552022 BBa_K3552023 BBa_K3552024 BBa_K3552025 BBa_K3552026 BBa_K3552027 BBa_K3552028 BBa_K3552029. Parts that are a complete circuit: BBa_K3552009 BBa_K3552010 BBa_K3552011 BBa_K3552012.

Our part collection can instruct other teams to designed new rechargeable pilus and substitution of different major pilin.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

GsPilA are fimbrial protein which normally grow on Geobacter sulfurreducens. They are type 4 pili which are long and thin which display on the cell surface membrane. They can promote adherence, motility and transport functions in the bacteria. They are mainly built as helical polymers of a single subunit called the major pilin. These pilins are initially in the plasma membrane and the N-terminal is positively charged. The mature pilins will be extracted through the membrane by the pilin generator.

Characterization

We first constructed the GsPilA by SOE PCR through 8 different primers to maintain the exactly same amino acid sequence as in the Geobacter sulfurreducens. After the final construction of the generator of PilA, we added six His-tag to the GsPilA and expressed the gene in vivo in E.coli BL21. After 48 hours of growth, we extracted the pili and verified the existence of GsPilA on the outer cell through Western Blot. We also do electronic speculum and see the clear image of the pili in the solution.

To get a complete pili production system, we first ordered oligo DNAs from Gs pilA gene and assembled them by SOE PCR to acquired its completely same amino acid sequence as expressed by EHEC genes. Then we constructed the generator of the pili and obtain a complete circuit of BBa_K3552010 .

For pili expression, we chose E.coli BL21 because it is the best fit to our project as it has the highest pili yield among them and is easy to obtain. On the cultivation part, we cultured the bacteria in solid M9 medium. Additionally, we provide the bacteria with glycerol as the carbon source because it was confirmed to help improve the conductivity of the pili produced.

We transformed BBa_K3552010 into E.coli BL21. The bacteria was first cultivated on a LB medium plate with kanamycin at 30°C for 24h. Then, we scraped the E.coli off the LB plates with 6ml liquid M9 solution and then coated the collected bacteria solution on 20 M9 medium plates with IPTG and kanamycin. Finally, the 20 coated M9 plates were cultured at 30°C for 48h.

We harvested the bacteria from M9 plates after 48h cultivation by scrapping them off again with liquid M9 solution and collect the bacteria solution. By conducting the extraction and purification on the samples, we managed to get an amount Gs pili solution dissolving in 150mM ethanol-amine after filtration with a 100kDa membrane in nitrogen gas.

We also ordered a SEM Photography for our pili on the photographs, we can see that the pili was distributed evenly on the surface in porous structures between each pili individuals. In order to confirm the E.coli produced the correct e-pili, we add 6xHis-tags to the end of the Gs pilA gene sequence, and constructed a new plasmid. And the addition of His-tags will not affect the conductivity and electricity generation of the e-pili produced. Then, we conducted Western Blot experiment to convince the production. According to the positive strands on the membrane, Pili was confirmed to be produced and purified successfully, with an expected molecular mass of 10kDa. We also conducted experiments to measure the voltages of each batteries. First we manufactured six standard electrodes by using three pili, two pieces each, all with triple layer of pili covered. Then we measured the voltage and compared the result which shows that Gm pili has the highest value of measured voltage where Gs pili has the least.

In order to compare the pili yield, we established a new measurement for a quicker, clearer, and more accurate determination of pili production. We use his-tags antibodies to attach on the his-tags on the pili, and then the secondary antibodies will attached to the his-tags antibodies for coloration directly on the outer membrane of the bacteria. We analyse the datas and included the effects of two variables on pili production: cultivation time and mobile oxygen presence. The result shows a ranking from Gs pili to Pa pili to Gm pili.