Difference between revisions of "Part:BBa K3815001"
| (23 intermediate revisions by 2 users not shown) | |||
| Line 2: | Line 2: | ||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K3815001 short</partinfo> | <partinfo>BBa_K3815001 short</partinfo> | ||
| − | == | + | ==Description of this part== |
| − | + | ||
<h3><font size="3">Targeted protein</font> </h3> | <h3><font size="3">Targeted protein</font> </h3> | ||
| − | This part is for the | + | This part is for the purification of the antimicrobial peptide, CecropinA. This is derived from <i>Hyalophora cecropia</i>. This can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.<br> |
| + | |||
<h3><font size="3">Purification system</font> </h3> | <h3><font size="3">Purification system</font> </h3> | ||
| − | In order to purify the peptide, we adopted ELK16 system that the past iGEM teams had not used. This part is composed of ELK16, Mxe | + | [[File:ELK16.png|300px|right|thumb|Fig1.The mechanism of ELK16]] |
| + | In order to purify the peptide, we adopted ELK16 system that the past iGEM teams had not used. This part is composed of ELK16, Mxe GyrA intein, and PT linker. When this fused protein is produced, ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we can get the targeted protein.<br> | ||
| Line 18: | Line 20: | ||
==Purification== | ==Purification== | ||
| − | [[File:Engineering 203 SDS-PAGE①.png|300px|thumb|right| | + | [[File:Engineering 203 SDS-PAGE①.png|300px|thumb|right|Fig2. SDS-PAGE of purified peptide ]] |
<h3><font size="4.5">Expression</font> </h3> | <h3><font size="4.5">Expression</font> </h3> | ||
<ul> | <ul> | ||
<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm. | <li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm. | ||
| − | <li>when the OD exceeded 0.35, IPTG 2ml was added to induce the peptide expression. | + | <li>when the OD exceeded 0.35, 1 M IPTG 2ml was added to induce the peptide expression. |
| + | <li>Incubate at 30℃ at 180rpm for 6 hours after adding IPTG. | ||
</ul> | </ul> | ||
<h3><font size="4.5">Purification </font></h3> | <h3><font size="4.5">Purification </font></h3> | ||
1.When this fused protein were produced, it self-assembled and precipitated<br> | 1.When this fused protein were produced, it self-assembled and precipitated<br> | ||
2.The aggregate was collected by centrifugation.<br> | 2.The aggregate was collected by centrifugation.<br> | ||
| − | 3.Adding DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.<br> | + | 3.Adding 40mM DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.<br> |
4.SDSPAGE was performed in order to confirm the presence of it. | 4.SDSPAGE was performed in order to confirm the presence of it. | ||
<br> | <br> | ||
<br> | <br> | ||
Fig1 shows the result of SDS-PAGE. | Fig1 shows the result of SDS-PAGE. | ||
| − | The lane 1, 5,and 9 are the result of CecropinA.<br> CecropinA is 4051Da, so these date shows that we could not | + | The lane 1, 5,and 9 are the result of CecropinA.<br> CecropinA is 4051Da, so these date shows that we could not confirm its production. |
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3815001 parameters</partinfo> | <partinfo>BBa_K3815001 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| + | |||
| + | ==Reference== | ||
| + | 1.Steiner, H., Andreu, D., and Merrifield, R.B. (1988). Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects. Biochim. Biophys. Acta 939, 260–266. | ||
Latest revision as of 01:58, 22 October 2021
CecropinA-Mxe GryA intein-PT-linker-ELK16
Description of this part
Targeted protein
This part is for the purification of the antimicrobial peptide, CecropinA. This is derived from Hyalophora cecropia. This can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.
Purification system
In order to purify the peptide, we adopted ELK16 system that the past iGEM teams had not used. This part is composed of ELK16, Mxe GyrA intein, and PT linker. When this fused protein is produced, ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we can get the targeted protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 814
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 67
Illegal AgeI site found at 346 - 1000COMPATIBLE WITH RFC[1000]
Purification
Expression
- Cells were grown in 1000ml LB media at 37oC shaking at 180 rpm.
- when the OD exceeded 0.35, 1 M IPTG 2ml was added to induce the peptide expression.
- Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.
Purification
1.When this fused protein were produced, it self-assembled and precipitated
2.The aggregate was collected by centrifugation.
3.Adding 40mM DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.
4.SDSPAGE was performed in order to confirm the presence of it.
Fig1 shows the result of SDS-PAGE.
The lane 1, 5,and 9 are the result of CecropinA.
CecropinA is 4051Da, so these date shows that we could not confirm its production.
Reference
1.Steiner, H., Andreu, D., and Merrifield, R.B. (1988). Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects. Biochim. Biophys. Acta 939, 260–266.