Peter E Burby —BIO-PROTOCOL

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Lyle Simmons Department of Molecular, Cellular, and Developmental Biology, Univerisity of Michigan, United States of America
1 protocol

David Cisneros Umeå University
33 protocols

Juan Facundo Ayala Instituto de Nanociencia y Nanotecnología
20 protocols

Marisa Rosa Bio-protocol.org
70 protocols

Rainer Melzer University College Dublin
13 protocols

Reviewer

Peter E Burby

Graduate, Univerisity of Michigan

Research focus

Microbiology
DNA damage response, DNA repair, stress response

1 Author merit
Reviewer merit
Editor merit

Education

Bachelor's of Science, Univerisity of Michigan, 2012

Lab information

Lyle Simmons Lab
https://sites.lsa.umich.edu/simmons-lab/

Publications

https://www.ncbi.nlm.nih.gov/pubmed/?term=Burby+PE

1 Protocol published

CRISPR/Cas9 Editing of the Bacillus subtilis Genome

Authors: Peter E. Burby and Lyle A. Simmons, date: 04/20/2017, view: 14177, Q&A: 4

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 ...

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Raquel Azevedo

Hi Mr. Burby,

First, I would like to thanks you for this very helpful protocol. I am now doing the Gibson reaction and transformation of MC1061 competent cells. I get colonies after transformation but with no way to check if the plasmid was correctly assembled and if it is in. I tried colony PCR, miniprep followed by PCR or send for sequencing (low yield). None of these worked. Actually, I get a low yield of the plasmid after miniprep. Is that normal? I'm using a miniprep kit from qiagen, do you use a different kit? or trick for the plasmid extraction?
My other question regards the transformation of the Bacillus strains. I'm working with environmental isolates, mainly B. licheniformis. Did you ever tried using this method in other strains other than B. subtilis? Maybe you could give me some feedback.

Many thanks for your time and help,
Kind regards,
Raquel

2/20/2019 2:15:57 AM Reply

Tobias Zumbusch

Fachhochschule Aachen

Hello Raquel,

how many clones did you pick for screening regarding the spacer integration?

As soon as i remember it's "low-copy" plasmid meaning you will only get around 50 ng/ul plasmid for a standard plasmid mini prep kit. so instead of using just 2 ml try to use 4ml ore more for the initial cell harvest, perform a bit variation regarding lysozyme and use for the final elution step not 50 ul maybe 30 ul. That will be still enough for further cloning procedures.

I'm also focussing on B. licheniformis but since i purified more than 100 clones and spacer integration was never succesful i switched to another plasmid.

Best
Tobias Zumbusch

2019-07-05 02:08:33

3 Protocols reviewed

Efficient Assembly of Large Multiplex CRISPR/Cas9 Guide Arrays for Maize Genome Editing

Authors: Jarrett Man and Madelaine E. Bartlett, date: 05/05/2019, view: 3082, Q&A: 0

CRISPR/Cas9-based genome editing in maize is an effective tool, and researchers commonly wish to target multiple genes simultaneously. Transformation of maize is currently expensive and tedious, so researchers are incentivized to build vectors that ...

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CRISPR/Cas9-mediated ssDNA Recombineering in Corynebacterium glutamicum

Authors: Jiao Liu, Yu Wang, Ping Zheng and Jibin Sun, date: 10/05/2018, view: 3879, Q&A: 0

Corynebacterium glutamicum is a versatile workhorse for industrial bioproduction of many kinds of chemicals and fuels, notably amino acids. Development of advanced genetic engineering tools is urgently demanded for systems metabolic ...

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