Part:BBa_K2560001:Experience

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Experiences of working with the Marburg Collection in E. coli

We, the iGEM Team Heidelberg 2010 really liked working with the Marburg Collection in Golden Gate Cloning and want to thank the iGEM team Marburg 2018 for creating such a great library of biobricks. We successfully showed that the Marburg Collection can be used in E. coli as well. For our work, we adapted the protocols of the iGEM Marburg Team 2018 (especially in the colony PCR) and developed a new protocol for another ligase, the NEB Hi-T4 Ligase.

Futur iGEM teams we want to give the following tips:

A lot of thoughts should be put into the primer design.

In Level 0 cloning, only 7.5 ng of the entry vector should be used.

The resistance gene should always be added in a molar concentration 1/10-fold compared to the other components in Level 1 and 2 cloning.

Cells do not have to be lysed for colony PCRs.

Loooooots of colonies should be picked (Yey picking parties!).
For more information about this, please check out our wiki.

Applications of BBa_K2560001

In our project we used this as a vector for using Golden Gate cloning to build our construct. The part was synthesized with added RFC10 prefix and suffix and then ligated to pSB1C3 according to the RFC10 standard. The vector plasmid was then purified and used in Golden Gate cloning. The new constructs made using this vector were then transformed into TOP10 cells which were grown on LB agar plates. The red fluorescence dropout colonies were easy to see by visual inspection but required over 24 hours after transformation for the RFP to be visible to the naked eye. The plates could be put in cold storage (4 °C) after 16 hours without any noticeable delay of the colonies becoming red [Aalto-Helsinki 2020].

User Reviews

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