Difference between revisions of "Part:BBa K5301005"

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==Cultivation, Purification and SDS-PAGE==
 
==Cultivation, Purification and SDS-PAGE==
 
===Induction===
 
===Induction===
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<h3>Results</h3>
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<p>Firstly, plasmids contains modified PL-promoter were constructed through PCR. During the amplification, The plasmid was divided into two parts (2.2 kb and 2.1 kb respectively) by 2 pairs of primers, and the results of agarose gel electrophoresis were shown below (Fig 1). The right position of sample DNA between makers indicated the success ligation of our PL. </p>
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                <b>Figure 1. PL ligation through PCR.</b>
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    <figcaption>Figure 1 | SDS-PAGE analysis of SCSdC-mCh[1-10] protein with IPTG concentration gradient induction. An IPTG concentration of 0.8mM was used for 16 hours induction at 16°C.</figcaption>
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    <p>Basic Silinker (BS) is a novel recombinant protein that efficiently attaches to the surface of silicon dioxide. The presence of mSA often leads to the formation of inclusion bodies, increasing the difficulty in purification. To achieve efficient expression of Basic Silinker and minimize the formation of inclusion bodies, we screened the induction conditions using IPTG. We set up a gradient of five IPTG concentrations: 0mM, 0.1mM, 0.25mM, 0.5mM, and 1mM. The results showed that the optimal protein expression was achieved with a concentration of 1mM. Furthermore, we tested two temperature gradients for induction: 37°C and 16°C. At 37°C, the protein mainly formed inclusion bodies rather than soluble proteins. Therefore, we determined that 16°C was the effective induction temperature.
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To facilitate proper folding of mSA and reduce inclusion body formation, we modified the protein buffer by incorporating biotin. The binding of biotin to mSA helps with the correct folding of the Basic Silinker protein, minimizing the formation of misfolded inclusion bodies. As a result, we obtained soluble proteins that could be extracted from the supernatant. The formulation of the buffer and experimental procedures can be found in our protocol.</p>
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Revision as of 17:16, 30 September 2024


SCSdC-mCh[1-10] is one of the components of multi-polymerized MSP.

Usage and Biology

In order to produce large nanodiscs more conveniently, we hope to flexibly extend the length of MSP according to demand, and thus propose the concept of multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MSP fragments. We used NW15 as the basic MSP and selected three types of linkers (Spy/Sdy/Snoop) to achieve the connection of different MSP fragments through the formation of covalent bonds, and adopted rigorous design to prevent self-cyclization of each fragment of the multi-polymer MSP. Finally, the successful cyclization of large circular MSPs is characterized by the fluorescence of mCherry after the combination of mCherry [1-10] and mCherry [11]. SCSdC-mCh[1-10], the first component of multi-polymerized MSP, is a fusion protein composed of SpyCatcher, mCherry [1-10], NW15, and SdyCatcher, with flexible GS linkers used to connect each part.

Cultivation, Purification and SDS-PAGE

Induction

Results

Firstly, plasmids contains modified PL-promoter were constructed through PCR. During the amplification, The plasmid was divided into two parts (2.2 kb and 2.1 kb respectively) by 2 pairs of primers, and the results of agarose gel electrophoresis were shown below (Fig 1). The right position of sample DNA between makers indicated the success ligation of our PL. 

           <a href="bba-k4634018-2.jpg" class="image">
<img alt="" src="bba-k4634018-2.jpg" width="100%" height=auto class="thumbimage" /></a> 
                   <a href="bba-k4634018-2.jpg" class="internal" title="Enlarge"></a>

               Figure 1. PL ligation through PCR.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1720
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1239
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1035
    Illegal AgeI site found at 1828
  • 1000
    COMPATIBLE WITH RFC[1000]