Team:TUDelft/Conjugation Procedure

=Experimental Procedures=

This page contains the step-by-step plan followed by the conjugation team and each steps current status.

Section 1: The Plan
Part 1A:
 * Acquire R751 plasmid &#10004;
 * Confirm wild R751 conjugation &#10004;
 * Characterize conjugation efficiency &#10004;

Part 1B: oriTR knockout
 * Design and order primers needed for &lambda;-red knockout &#10004;
 * Acquire knockout plasmids &#10004;
 * Knockout oriTR **In Progress**
 * Verify that conjugation stopped **In Progress**
 * Characterize conjugation efficiency of Conjugation Testing Plasmid 1 with R751 &Delta;oriTR as helper
 * Send R751 &Delta;oriTR plasmid to registry

Part 1C: trbK knockout
 * Knockout trbK **In Progress**
 * Verify that conjugation takes place among R751 &Delta;trbK cells
 * Characterize conjugation efficiency
 * Send R751 &Delta;oriT + &Delta;trbK plasmid to registry

Part 1D: trbC knockout
 * Knockout trbC
 * Verify that no conjugation takes place in presence of Conjugation Testing Plasmid 1
 * Send R751 &Delta;oriT + &Delta;trbK + &Delta;trbC plasmid to registry

Knockouts
The &lambda;-red knockout system was selected as the procedure for doing the knockouts. It had previously been used by several people and demonstrated to work (lambda red, NanoBio, knockout protocol used by Berkeley '06). Primers were designed following the standard procedure. The following primers were used (red parts are P1 and P2):

trbK_KO_FWD (70 bp)

CCAGGGCAGCTACCGGGCCAGCCCGGCGCGCACCTGGTAAGGGGGGATTC GTGTAGGCTGGAGCTGCTTC

trbK_KO_REV (70 bp)

GCGGCAGGGCGAGGGTTTTTAGATTGGCTGGCATTCTCATCGTCAGCACC ATGGGAATTAGCCATGGTCC

oriTR_KO_FWD (70 bp)

TCGCGCAGATAGCGCGCCACGCTGACGCCCGCCCTCTTGGCGTTCGCCTC GTGTAGGCTGGAGCTGCTTC

oriTR_KO_REV (70 bp)

TTTCGCTATATCCGTTGCTGCTTTTGCGGCCTGATAGCGCGATAGTTGCG ATGGGAATTAGCCATGGTCC

The following verification primers were used (blue portions are on R751 outside the 50 bp upstream region):

trbK_KO_FWD (20 bp)

CACAACTGCG CCAGGGCAGC

trbK_KO_REV (20 bp)

CAGACGAACA GCGGCAGGGC

oriTR_KO_FWD (20 bp)

CTGGCCCACG TCGCGCAGAT

oriTR_KO_REV (20 bp)

TTGTGGCGGG TTTCGCTATA

Linear fragments were created using pKD4 (KAN) as a template using Platinum® Pfx DNA Polymerase. See Results page for more info.

Section 2: Message Plasmid
Part 2A: BioBrick Assembly
 * Order DNA synthesis for
 * BBa_K175001 (trbK) &#10004;
 * BBa_K175000 (trbC)
 * Verify that trbK expression blocks conjugation &#10004;
 * Place trbK on standard backbone &#10004;, sequence &#10004;, and send to registry &#10004;
 * Amplify and Transform BioBricks needed
 * BBa_I13522 (pTet-RBS-GFP-term-term) &#10004;
 * BBa_I714031 (oriTR) &#10004;
 * BBa_J13002 (pTet-RBS) &#10004;
 * BBa_E0840 (RBS-GFP-term-term) &#10004;
 * Assemble Conjugation Testing Plasmid 1: [promoter][GFP generator][oriT] &#10004;
 * Verify Conjugation Testing Plasmid 1 works. &#10004;
 * Sequence Conjugation Testing Plasmid 1. **In Progress**
 * Assemble Conjugation Testing Plasmid 2 [promoter][rbs][trbK][rbs][GFP][oriT] &#10004;
 * Verify Conjugation Testing Plasmid 2 works. **In Progress**
 * Sequence Conjugation Testing Plasmid 2. **In Progress**

Part 2B: Full Communication testing
 * Electroporate Conjugation Testing Plasmid 2 into some R751 &Delta;oriT cells creating InitiatorCells (select for presence of both message and helper plasmid)
 * Add InitiatorCells to a culture of R751 &Delta;oriT + &Delta;trbK and observe signal propagation, characterize rate of signal propagation. Look for lethal zygosis issues.
 * If signal propagation observed, do victory dance.

For more information on the cloning strategy of constructing the conjugation plasmids and knockouts check the cloning strategy page

On to the Experimental Results >>>