Difference between revisions of "Part:BBa K3888006:Experience"

 
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
−
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
−
how you used this part and how it worked out.
 
−
 
 
===Applications of BBa_K3888006===
 
===Applications of BBa_K3888006===
 +
In order to achieve gene knockout, we used pK18mobsacB-Gm plasmid as a knockout vector. In addition, in order to achieve knockout, we selected about 1000bp sequences in the upstream and downstream of UppE and HupS respectively for design, amplified from the Rhodopseudomonas palustris CGA009 by PCR, and constructed knockout plasmids by the Gibson method (see the Design section for principles) ). The results of plasmid design and construction are as follows:
 +
[[File:UppE results.png]]<br>
 +
===Figure 1. Plasmid Design and Agarose Gel Result. Left: UppE Plasmid Design. Middle: HupS Plasmid Design. Right: Agarose gel result (0.8%).===
 +
After the plasmid construction is completed, we use the method of electrotransformation to transfer the plasmid into the cell. Afterwards, colonies in which the plasmid was successfully integrated into the chromosome were screened by the gentamicin resistance gene on the vector. After that, the medium containing 10% sucrose was used for secondary screening to facilitate the excision of the inserted plasmid sequence in the double exchange event. Finally, the selected individuals are amplified and cultured, and the genome is extracted and verified by PCR. In order to advance the progress of the experiment, the first round of knockout experiments were carried out separately, and the knockout results are as follows:
 +
[[File:UppE results 2.png]]<br>
 +
===Figure 2. Gene knockout design.===
 +
The knockout screening of UppE was not successful. In the end, we failed to complete the knockout of UppE.
 +
===User Reviews===
  
−
===User Reviews===
 
 
<!-- DON'T DELETE --><partinfo>BBa_K3888006 StartReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K3888006 StartReviews</partinfo>
−
<!-- Template for a user review
+
Reference:
−
{|width='80%' style='border:1px solid gray'
+
1. Fritts, Ryan K., et al. "A Rhizobiales-specific unipolar polysaccharide adhesin contributes to Rhodopseudomonas palustris biofilm formation across diverse photoheterotrophic conditions."Applied and environmental microbiology 83.4 (2017): e03035-16<!-- End of the user review template -->
−
|-
+
−
|width='10%'|
+
−
<partinfo>BBa_K3888006 AddReview number</partinfo>
+
−
<I>Username</I>
+
−
|width='60%' valign='top'|
+
−
Enter the review inofrmation here.
+
−
|};
+
−
<!-- End of the user review template -->
+
 
<!-- DON'T DELETE --><partinfo>BBa_K3888006 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K3888006 EndReviews</partinfo>

Revision as of 15:34, 20 October 2021


Applications of BBa_K3888006

In order to achieve gene knockout, we used pK18mobsacB-Gm plasmid as a knockout vector. In addition, in order to achieve knockout, we selected about 1000bp sequences in the upstream and downstream of UppE and HupS respectively for design, amplified from the Rhodopseudomonas palustris CGA009 by PCR, and constructed knockout plasmids by the Gibson method (see the Design section for principles) ). The results of plasmid design and construction are as follows: UppE results.png

Figure 1. Plasmid Design and Agarose Gel Result. Left: UppE Plasmid Design. Middle: HupS Plasmid Design. Right: Agarose gel result (0.8%).

After the plasmid construction is completed, we use the method of electrotransformation to transfer the plasmid into the cell. Afterwards, colonies in which the plasmid was successfully integrated into the chromosome were screened by the gentamicin resistance gene on the vector. After that, the medium containing 10% sucrose was used for secondary screening to facilitate the excision of the inserted plasmid sequence in the double exchange event. Finally, the selected individuals are amplified and cultured, and the genome is extracted and verified by PCR. In order to advance the progress of the experiment, the first round of knockout experiments were carried out separately, and the knockout results are as follows: UppE results 2.png

Figure 2. Gene knockout design.

The knockout screening of UppE was not successful. In the end, we failed to complete the knockout of UppE.

User Reviews

UNIQ80a5f1879e869d2d-partinfo-00000000-QINU Reference: 1. Fritts, Ryan K., et al. "A Rhizobiales-specific unipolar polysaccharide adhesin contributes to Rhodopseudomonas palustris biofilm formation across diverse photoheterotrophic conditions."Applied and environmental microbiology 83.4 (2017): e03035-16 UNIQ80a5f1879e869d2d-partinfo-00000001-QINU