Difference between revisions of "Team:UGent Belgium/LabNotebook"
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| − | We transformed E. coli TOP10 cells with our weak expression vector (pXW), which came in today. Q5 CPEC was used for this with the linearized plasmid backbone pSB1C3 and as insert a gBlock consisting of promoter-RBS-cloning site-terminator-terminator. The promoter is weaker than the promoter of the strong expression vector (cf. 29/08). | + | We transformed <i>E. coli</i> TOP10 cells with our weak expression vector (pXW), which came in today. Q5 CPEC was used for this with the linearized plasmid backbone pSB1C3 and as insert a gBlock consisting of promoter-RBS-cloning site-terminator-terminator. The promoter is weaker than the promoter of the strong expression vector (cf. 29/08). |
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</ul> | </ul> | ||
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| + | |||
| + | <li class="list-group-item borderless"><b>September 06:</b> <p> | ||
| + | Colonies from our strong expression vector made with CPEC (cf. 04/09) are cultured and miniprepped. These are used for following constructs: | ||
| + | <ul> | ||
| + | <li>pXS-INP_WT using the inaZ PCR fragment </li> | ||
| + | <li>pXS-INP_NC-mGFPuv </li> | ||
| + | <li>pXS-INP_NC-Strep (Strep: regular streptavidin)</li> | ||
| + | <li>pXS-INP_NC-mSA2 (mSA2: monomeric streptavidin)</li> | ||
| + | <li>pXS-Lpp-ompA-mGFPuv </li> | ||
| + | <li>pXS-Lpp-ompA-Strep</li> | ||
| + | <li>pXS-Lpp-ompA-mSA2 </li> | ||
| + | </ul> | ||
| + | All constructs are made by using a Golden Gate reaction mix | ||
| + | </li> | ||
</ul> | </ul> | ||
Revision as of 21:00, 6 October 2016
Lab Notebook
- August 29:
- We made a strong expression vector (pXS) by restriction of the linearized plasmid backbone pSB1C3 and insertion of gblock made up of promoter-RBS-cloning site-terminator-terminator (cut with EcoRI and PstI).
- Our first attempts to isolate the inaZ gene from P. syringae and the inaX gene from X. campestris failed.
- August 30:
- We transformed the strong expression vector (cf. 29/08) by electroshock into E. coli TOP10.
- As a backup strategy we also used CPEC with the backbone from the K584027 plasmid to generate a strong expression vector.
- August 31:
- Second attempt to isolate the inaZ gene from P. syringae and the inaX gene from X. campestris. InaX failed, but inaZ was successful.
- Transformation of the strong expression vector by restriction and ligation was not very successful, we only had a few colonies. The backbone amplification using CPEC was successful (cf. 30/08).
- September 01:
- Heatshock transformation of the strong expression vector made with CPEC yielded no colonies.
- Colony PCR on the colonies with the strong expression vector made with restriction and ligation didn’t show any positive ones (cf. 31/08).
- September 02:
Electroshock transformation of the strong expression vector made with CPEC into E. coli TOP10 cells.
- September 04:
- Via colony PCR we saw that all colonies with the strong expression vector made with CPEC and electroshock transformation were all positive (cf. 02/09).
- We transformed E. coli TOP10 cells with our weak expression vector (pXW), which came in today. Q5 CPEC was used for this with the linearized plasmid backbone pSB1C3 and as insert a gBlock consisting of promoter-RBS-cloning site-terminator-terminator. The promoter is weaker than the promoter of the strong expression vector (cf. 29/08).
- September 06:
Colonies from our strong expression vector made with CPEC (cf. 04/09) are cultured and miniprepped. These are used for following constructs:
- pXS-INP_WT using the inaZ PCR fragment
- pXS-INP_NC-mGFPuv
- pXS-INP_NC-Strep (Strep: regular streptavidin)
- pXS-INP_NC-mSA2 (mSA2: monomeric streptavidin)
- pXS-Lpp-ompA-mGFPuv
- pXS-Lpp-ompA-Strep
- pXS-Lpp-ompA-mSA2
