Difference between revisions of "Part:BBa K3552000"

(Characterization)
 
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<partinfo>BBa_K3552000 short</partinfo>
 
<partinfo>BBa_K3552000 short</partinfo>
  
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.
+
Geobacter sulfurreducens pilA(GsPilA) is a type 4 pilus usually found 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 of stability and yield.
  
 
The part collection includes:
 
The part collection includes:
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<partinfo>BBa_K3552012</partinfo>.
 
<partinfo>BBa_K3552012</partinfo>.
  
Our part collection can instruct other teams to designed new rechargeable pilus and substitution of different major pilin.
+
Our part collection can instruct other teams to design new rechargeable pilus and substitute them to have more combinations that may achieve better performance in the future when manufacturing the batteries.
  
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<span class='h3bb'>'''Sequence and Features'''</span>
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==Usage and Biology==
 
==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.
+
GsPilA is a fimbrial protein that normally functions in Geobacter sulfurreducens which can conduct electricity. It is a type 4 pili that is long and thin, displaying on the cell surface membrane. It can promote adherence, motility, and transport functions in the bacteria. It is 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 can be extracted through the membrane by the pilin generator.
  
 
==Characterization==
 
==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.
 
  
          [[File:T--LINKS China--Figure 1.jpeg|800px]]
+
===Construction===
 +
This year, LINKS_China constructed a conductive pilus, GsPilA, through slicing by overlap extension by PCR using 8 different primers to obtain a similar DNA sequence as in the Geobacter sulfurreducens, which produce the same amino acid sequence. To get a complete pili production system, we constructed the generator of the pili and obtained 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> .
+
        [[File:T--LINKS China--Figure pili expression.jpeg|800px]]
  
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.
+
        [[File:T--LINKS China--Figure his-tag addition.jpeg|800px]]
  
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.
+
For pili expression, we chose E.coli BL21 because it is the best fit for our project as it has the highest pili yield and is easy to obtain. On the cultivation part, we cultured the bacteria in solid M9 mediums. Additionally, we provided the bacteria with glycerol as the carbon source because it was confirmed to help to improve the conductivity of the pili produced.
  
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.  
+
===Protein production===
 +
Transformed <partinfo>BBa_K3552010</partinfo>  into Escherichia coli(E.coli) BL21, we cultivated the bacteria and harvested the bacteria from M9 plates after 48h cultivation by scrapping them off again with liquid M9 solution and collect the bacteria solution. Finally, by conducting the extraction and purification of 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.
+
        [[File:T--LINKS China--Figure pilin production.jpeg|800px]]
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.
+
We also ordered an SEM Photography for our pili on the photographs, we can see that the pili were distributed evenly on the surface in porous structures between each pili individuals. To confirm the E.coli produced the correct e-pili, we conducted a Western Blot experiment to convince the production. According to the positive strands on the membrane, we confirmed pili, with an expected molecular mass of 10kDa, was produced and purified successfully.
 +
 
 +
        [[File:T--LINKS China--Figure expression in E.coli.jpeg|800px]]
 +
 
 +
===Conductiviity measure===
 +
We manufactured the extracted pili into biological batteries and we also conducted experiments to measure the voltage of each battery. First, we manufactured six standard electrodes by using three pili, two pieces each, all with a triple layer of pili covered. Then we measured the voltage and compared the result which showed that Gm pili have the highest value of measured voltage while Gs pili have the least.
 +
 
 +
        [[File:T--LINKS China--Figure battery.jpeg|800px]]
 +
 
 +
===New measurement===
 +
To compare the pili yield, we established a new measurement for a quicker, clearer, and more accurate determination of pili production. We used his-tags antibodies to attach to the his-tags on the pili, and then the secondary antibodies will be attached to the his-tags antibodies for coloration directly on the outer membrane of the bacteria. We analyzed the data and included the effects of two variables on pili production: cultivation time and mobile oxygen presence.
 +
 
 +
        [[File:T--LINKS China--Figure verification.jpeg|800px]]
 +
 
 +
        [[File:T--LINKS China--Figure results of new measurement.jpeg|800px]]
 +
 
 +
We measured the absorbance of all samples at od 450 divided by od 600 and subtract the value of negative control to obtain optimized od 450. The result shows a ranking of yield from the highest Gs pili to the lowest Gm pili. The trend for three experiments shows a smaller error bar for expression with EHEC similar generator. The sealed plates also received better results of higher production than the unsealed ones. The optimum cultivation time for GsPilA is 48 hours as it has an optimized od 450 value of 0.4 higher than 72 hours cultivation, whereas at 24 hours cultivation all kinds of bacteria expressed pili poorly.
  
 
<partinfo>BBa_K3552000 parameters</partinfo>
 
<partinfo>BBa_K3552000 parameters</partinfo>
 
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Latest revision as of 12:42, 27 October 2020


GsPilA

Geobacter sulfurreducens pilA(GsPilA) is a type 4 pilus usually found 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 of stability and yield.

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 design new rechargeable pilus and substitute them to have more combinations that may achieve better performance in the future when manufacturing the batteries.

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]

Reference

Luna Rico, Areli et al. “Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli.” Molecular microbiology vol. 111,3 (2019): 732-749. doi:10.1111/mmi.14188

Usage and Biology

GsPilA is a fimbrial protein that normally functions in Geobacter sulfurreducens which can conduct electricity. It is a type 4 pili that is long and thin, displaying on the cell surface membrane. It can promote adherence, motility, and transport functions in the bacteria. It is 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 can be extracted through the membrane by the pilin generator.

Characterization

Construction

This year, LINKS_China constructed a conductive pilus, GsPilA, through slicing by overlap extension by PCR using 8 different primers to obtain a similar DNA sequence as in the Geobacter sulfurreducens, which produce the same amino acid sequence. To get a complete pili production system, we constructed the generator of the pili and obtained a complete circuit of BBa_K3552010 .

        T--LINKS China--Figure pili expression.jpeg
        T--LINKS China--Figure his-tag addition.jpeg

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

Protein production

Transformed BBa_K3552010 into Escherichia coli(E.coli) BL21, we cultivated the bacteria and harvested the bacteria from M9 plates after 48h cultivation by scrapping them off again with liquid M9 solution and collect the bacteria solution. Finally, by conducting the extraction and purification of 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.

        T--LINKS China--Figure pilin production.jpeg

We also ordered an SEM Photography for our pili on the photographs, we can see that the pili were distributed evenly on the surface in porous structures between each pili individuals. To confirm the E.coli produced the correct e-pili, we conducted a Western Blot experiment to convince the production. According to the positive strands on the membrane, we confirmed pili, with an expected molecular mass of 10kDa, was produced and purified successfully.

        T--LINKS China--Figure expression in E.coli.jpeg

Conductiviity measure

We manufactured the extracted pili into biological batteries and we also conducted experiments to measure the voltage of each battery. First, we manufactured six standard electrodes by using three pili, two pieces each, all with a triple layer of pili covered. Then we measured the voltage and compared the result which showed that Gm pili have the highest value of measured voltage while Gs pili have the least.

        T--LINKS China--Figure battery.jpeg

New measurement

To compare the pili yield, we established a new measurement for a quicker, clearer, and more accurate determination of pili production. We used his-tags antibodies to attach to the his-tags on the pili, and then the secondary antibodies will be attached to the his-tags antibodies for coloration directly on the outer membrane of the bacteria. We analyzed the data and included the effects of two variables on pili production: cultivation time and mobile oxygen presence.

        T--LINKS China--Figure verification.jpeg
        T--LINKS China--Figure results of new measurement.jpeg

We measured the absorbance of all samples at od 450 divided by od 600 and subtract the value of negative control to obtain optimized od 450. The result shows a ranking of yield from the highest Gs pili to the lowest Gm pili. The trend for three experiments shows a smaller error bar for expression with EHEC similar generator. The sealed plates also received better results of higher production than the unsealed ones. The optimum cultivation time for GsPilA is 48 hours as it has an optimized od 450 value of 0.4 higher than 72 hours cultivation, whereas at 24 hours cultivation all kinds of bacteria expressed pili poorly.