Difference between revisions of "Part:BBa K4214003"
Line 14: | Line 14: | ||
*The LacI promotor is described in part [https://parts.igem.org/Part:BBa_J04500 BBa_J04500] | *The LacI promotor is described in part [https://parts.igem.org/Part:BBa_J04500 BBa_J04500] | ||
− | |||
− | |||
− | |||
− | |||
<br><br><br> | <br><br><br> | ||
<br><br><br> | <br><br><br> | ||
<br><br><br> | <br><br><br> | ||
<br><br><br> | <br><br><br> | ||
+ | <br><br><br> | ||
+ | <br><br><br> | ||
+ | |||
+ | =Characterization= | ||
+ | <hr> | ||
+ | ===SDS-PAGE=== | ||
+ | A production culture was set up using BL21 ''E. coli'' and induced with 0.5 mM IPTG once their ODs reached 0.5. The cultures were left overnight at 30°C for protein production. The cells were then harvested, pelleted and purified using a HisPur Cobalt resin. The SDS-PAGE analysis of the production process is shown on the right. | ||
<br><br><br> | <br><br><br> | ||
<br><br><br> | <br><br><br> | ||
<br><br><br> | <br><br><br> | ||
<br><br><br> | <br><br><br> | ||
+ | |||
[[Image:BBa K4214003 SDSPAGE.png|600px|thumb|center|Fig.: ''SDS-PAGE of BBa K4214003 part for protein production'']] | [[Image:BBa K4214003 SDSPAGE.png|600px|thumb|center|Fig.: ''SDS-PAGE of BBa K4214003 part for protein production'']] |
Revision as of 13:26, 9 October 2022
hGIF with LacI promotor and 6xHis-tag
LacI (BBa_J04500) and Human GIF with His6-tag. This part is a composite part from BBa_K4214002 with the addition of LacI sequence (BBa_J04500). The LacI sequence is used in order to induce our protein expression with IPTG, and the 6xHis-tag was used for protein purification.
Usage and Biology
- NCBI page of the human gastric intrinsic factor [1]
- This part of hGIF does not contain the native signal peptide (AA1-18)
- The LacI promotor is described in part BBa_J04500
Characterization
SDS-PAGE
A production culture was set up using BL21 E. coli and induced with 0.5 mM IPTG once their ODs reached 0.5. The cultures were left overnight at 30°C for protein production. The cells were then harvested, pelleted and purified using a HisPur Cobalt resin. The SDS-PAGE analysis of the production process is shown on the right.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 485
Illegal BsaI.rc site found at 1165
Illegal SapI site found at 1155