# Presentation: 144508

## Deletion mutageneis of Desulfovibrio vulgaris using advanced PCR methods 

**Deletion mutageneis of *****Desulfovibrio vulgaris***** using advanced PCR methods **

**B.M. Emo, E. Drury, and J.D Wall**

- Department of Biochemistry         117 Schweitzer Hall                  University of Missouri - Columbia Columbia, MO,  65211 emob@missouri.edu

_**PCR Junction Product**_

- .../

- /...

**PCR amplification**

**Hybridize**

_**PCR Product Description**_
**PCR1** has the KanR marker for exchange, molecular barcodes, and common sequence for monitoring mutants
**PCR2** has sequence upstream of target gene, ATG of target, and common sequences
**PCR3** has common sequences, stop codon of target gene, and sequence downstream of target
**PCR4** has three preceding products joined PCR2/1/3

_**PCR1**_
- Using pSC27 (Tn5 KanR, mob, pBG1) as a template, amplify the KanR cassette with 65-mer primers that add the 20 base barcode sequence and the 26 base common sequence.

_**PCR2 and PCR3**_
- Primers amplify the upstream and downstream sequences and add the common sequences proximal (with respect to the target sequence) and restriction sites distal.

_**PCR4**_
- By combining the three previous PCR product and the two outside primers, a continuous product is generated.

**PCR2 and PCR3 **(separate reactions)

**PCR4**

**ATG**

**TAA**

**Kan**

**Product of PCR2 (>630bp)**

**Product of PCR3 (>630bp)**

**Product of PCR1 (~1000bp)**

**PCR reactions have been  successfully tested (six deletion cassettes generated using this method).**
**The polymerase Herculase (Stratagene, taq/pfu mix) works well for generating all PCR fragments with a degree of fidelity.**
**Deletion cassettes successfully cloned into the TOPO cloning vector for storage.**
**Deletion cassettes reamplified and cloned into the mutagenic vector (pMO510 – map unavailable).**
**Electro-transformation is still a limitation for efficient generation of recombinants using ampicillin and chloramphenicol selection.**

**Molecular barcodes allow for the comparison of mutant and wild type fitness when in grown in co-culture.  Therefore the relative health (presence in culture) of any mutant compared to wild type correlates to the importance of the mutant gene in growth or stress response. **

**R1**

**Choose**
**Marker**

**Obtain**
**Sequence**

**Design **
**Primers**

**Order **
**Primers**

**PCR1**

**Repeat PCR**

**D1**

**D2**

**Store at –20****o****C**

**Gel Purify**

**PCR4**

**D4**

**Gel Purify**

**Ligate into **
**Storage Vector**

**Ligate into **
**Mut. Vector**

**R2**

**Choose Gene**
**for Deletion**

**Obtain Up/Down-**
**Stream Seq.**

**Design **
**Primers**

**Order **
**Primers**

**PCR2 and PCR3**

**Gel Purify**

**D3**

**Repeat PCR4**

**Change PCR**
**Conditions**

**Close**
**Book**

**B**

**Barcode**

**Common**

**Yes**

**No**

**No**

**No**

**No**

**Yes**

**No**

**Yes**

**Yes**

**B**

**B**

_**Southern Analysis of Integrates**_

- Southern analysis will be performed on genomic DNA isolated from each mutant and digested by an appropriate restriction endonuclease (considering the restriction sites contained in the deletion cassette).  Labeled probe is specific for the kanamycin resistance cassette and shifts in band size will be key in identifying single and double recombinants.

**Yes**

**No**

**Confirm Plasmid**
**Mutation**

**PCR Junction Product**

**Southern of**
**Digested Chromosome**

**R3**

**R2**

**Close**
**Book**

**D6**

**Purify Ab****1****R**** CFU**

**R2**

**D5**

**No**

**Yes**

**Electro-transformation**
**into *****D.vulgaris***

**Purify Ab****1****R**** CFU**

**Enrich Sucrose****R**

**Plate Sucrose****R**
**and Ab****1****R**

**Screen Plasmid Ab****2****S**

**Yes**

**No**

**Confirm Deletion**
**Mutation**

**PCR Junction Product**

**Southern of**
**Digested Chromosome**

**D7**

**R4**

**R3**

**Grow and**
**Make Glycerol Stock**

**Growth Curves:**
**Carbon sources**
**Stress responses**

**Ship to LBL for Biomics**

**Close**
**Book**

**B**

**B**

**Conjugation into SRB**

**Construct mutant: First Recombination for Plasmid Integration**
**Construct mutant: Second Recombination **
**Characterize Mutant **

**Is there *****E. coli***** contamination?**
**Is plasmid integrated as expected?**
**Is plasmid integrated as expected?**
**Post to Biofiles**

**D6**

**D5**

**B**

**D7**

**R2**

**R3**

**R4**

- The junction product can be amplified using the Kan (out) primers and the upstream and downstream genomic primers.  These products can then be sequenced to check for integration and verify the sequence of the flanking barcodes.

_**Recombination Event 1**_
- The first recombination event integrates the vector and forms an intermediate product that is ampicillin and kanamycin resistant and sucrose sensitive.

_**Recombination Event 2**_
- The second recombination event removes all vector sequence, the target gene, and the plasmid component of the junctions leaving an ampicillin sensitive, kanamycin and sucrose resistant deletion mutation.

- 

- 

**Recombination 1**

**Recombination 2**

- .../

**a’b’**

**c’d’**

**a b**

**c d**

- .../

**c**** ****d’**

**a’****b**

**c’****d**

**a**** ****b’**

- Vector sequence	             *D.vulgaris *sequence AmpicillinR cassette  *sacB* sequence

**a’ b’**

**a b**

**c**** ****d’**

**c’****d**

- 

- .../

- 26 base common sequence         20 base unique barcode          KanamycinR cassette        Target gene sequence

**1 day**

**7-14 days**

**1-2 days**

**2 days**

**Generate deletion mutants for the purpose of:**
**Comparing mutant and wild type growth to determine the contribution of each mutant gene to the overall fitness of *****D.vulgaris*****.**
**Comparing the stress response performance of deletion mutants to wild type *****D.vulgaris*****.**

**B**

**B**