Team 3 Notebook

Members

 * Andrew Saarni
 * Kai Mesa
 * Jose Gutierrez
 * Zhen Huang

First group meeting
General Project outline: Origin of Stability and copy number
 * Characterize the orthogonality of 3 colE2 replicons: CA42, 099, P9.
 * Quantify the regulatory effects of using the natural 5' regulatory elements of these replicons systems in E. coli.

Second Group Meeting
Worked on Project Presentation

Team 3

[ColE2 Ori / Rep System]

-Origin Stability and Copy Number-

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[Objective]

-Design assays to...


 * 1) Test the orthogonality and stability of regulation for 27 different ColE2 replicons, prepared by combining different Ori/Rep plasmids and promoter regions


 * 1) Determine the effect of regulatory elements on each plasmid with regard to controlling the copy number of the ColE2 Ori.

[ColE2 Ori / Rep System]

- Description

- ColE2 family of replicons is regulated by two elements:

- Element 1: ColE2 Origin of Replication

a.) Located on the plasmid (in cis) (1)

b.) Minimal required region for Rep binding is 31bp (2)

c.) 3 subregions: Regions I and II are essential for Rep

binding; Regions II and III are essential for replication (2)

- Element 2: Rep(lication) protein

a.) Expressed CDS located either in cis or in trans (1)

b.) Acts also as a plasmid-specific primase; synthesizes

3bp RNA primer used for polymerase binding  (2)

- The presence of these two elements allows the replication of

the circular plasmid DNA containing the ColE2 Ori. (1)

[ColE2 Ori / Rep System]

- Purpose and Regulation

- Allows implementation of conditionally active genes. (1)

- Transformed plasmid containing ColE2 Ori will remain

dormant until transcription of Rep protein is initiated. (1)

- Copy number of the transformed plasmid can therefore be

increased at will by enabling the ColE2 Ori. (1)

- In the case of the 5'UTR's, a regulatory RNA, which is

translated along with the rest of the ColE2 Ori in the

presence of Rep protein, provides negative translational

feedback by inhibiting translation of Rep protein.

[Our Plasmids]

All assembled parts are vectors with R6K ori

Rep  and Ori (CA42, 099, P9) are in separate plasmids

Using promoters Ptet and Pcon and b1006 terminator

Each plasmid will have a different resistance gene: Rep plasmids are AmpR and KanR, Ori plasmids are SpecR.

R6K ori requires additional gene called pir which encodes the pi protein to be functional and constant expression.

[Origin Stability Assay]

Motivation - Compatibility goups in a nutshell:

Two different plasmid with the same origin of replication in the same cell will compete for replication resources

One solution to the problem is to use two plasmids with different origins.

In this assay we want to transform cells with different ori/rep pairs out of CA42, 099, P9, and different promoters.

Assay shows where ori/rep pairs fall

equivalent «» orthogonal

(cross-reactive)                           (mutually independent)

[Two Step Transformation]

Problem: Cannot transform both Ori and Rep plasmids at the same time, because if they don't work we will not know if it was because the transformation failed or if parts are in fact orthogonal

Solution: two step transformation process


 * 1) Transform Rep plasmid into Bacteria (Ec100D)


 * 1) Plate on (Kan) plates, Pick Colonies


 * 1) Miniprep for plasmid DNA purification


 * 1) Analytical digest


 * 1) Once first transformation is done, make competent cells    using colony


 * 1) Repeat fist four steps with Ori Plasmid, Plate on (Kan/Spec) plates

[Plasmid Copy Number Assay]

Purpose:Determine the effect of regulatory elements on each plasmid with regard to controlling the copy number of the ColE2 Ori.


 * 1) need a way to standardize relative # of Ori plasmids/cell


 * 1) Rep plasid copy should be constant for each cell


 * 1) isolate both plasmids, and map


 * 1) We can correlate the highest number of Ori plasmids/cell by examining the relative intensities of the two bands

[Mapping Protocol]

Miniprep Purification of DNA

Analytical Digests (Mapping)

We will only use EcoR1 in our digests, since we only want to linearize the plasmids.

Note that the R6K and ori plasmids should be different in size by at least a few hundred base pairs, or it will be difficult to distinguish the plasmid bands on the gel.

[Regulatory Element Efficiency Assay]

Determine if a band at the ori plasmid size is present

If a band is present, this will suggest cross-reactivity between the ori/rep combination. And therefore are not orthogonal.

Note that the compliment combination of ori/rep could still be orthogonal.

[Orthogonality Assay]

If an ori plasmid band is detected, measure the relative band intensity, when compared to the correspondent R6K plasmid band.

The R6K plasmid should have a constant expression in all samples that can be used to relate the relative efficiency of each ori/rep combination.

[Different combinations]

For the Origin Stability Assay, our rep composite parts will use ptet and pcon promotors as well as a b1600 terminator

For the Plasmid Copy Number Assay, we will use the rep coding sequence along with its natural regulatory elements

For both assays, we will use the established colE2 replicon ori/rep pairs as positive controls.

All 27 combinations can be found on the Excel File

[References]

1. http://openwetware.org/wiki/Template:SBB10Project-27095

2.  PMID 17098894

Project Presentations
GoogleDoc Presentation

Eco/Bam Transfer
Ori basic parts need to be transfered into R6K vectors 6uL ddH2O 1 uL NEB2 Buffer 0.5uL EcoRI 0.5uL BamHI 2uL part plasmid Note: For R6K digest, we used 5μl of R6K plasmid and 3μl of ddH2O
 * We perfromed an Eco/Bam on parts: sbb24, sbb25, sbb26, sbb31 and sbb32 as well as an R6K vector.
 * Set up the digest:
 * Incubate for 1hr at 37°C
 * Zymo Cleanup
 * We used 10μl of ddH2O for the elution step in Zymo cleanup.

Digestion of Correct R6K Plasmid
The R6K plasmid we digested on 4/12/2010 did not have an EcoR1 site, so we digested a different R6K plasmid that did. 7uL ddH2O 2uL NEB2 Buffer 1uL EcoRI 1uL BamHI 9uL R6K plasmid
 * Set up the digest:
 * Incubate for 1hr at 37°C
 * Run out on gel
 * 1400bp band is our R6K vector
 * Cut out band and purify

Ligation Reaction
Ligation of ori basic parts into R6K vector
 * Ligation
 * Note: An extra ligation was performed with no insert (negative control)

Transformation
Transformation of ligation reactions into a pir+ strain
 * Transformation/Rescue/Plate
 * Note: LB Kan plates used.

Colony Inoculation

 * Colony Picking
 * Note: 2 colonies picked per plate

Miniprep

 * Miniprep
 * Note: 24.1, 24.2 (sbb24); 25.1, 25.2 (sbb25); 26.1, 26.2 (sbb26); 31.1, 31.2 (sbb31); 32.1, 32.2 (sbb32)

Digest

 * Digest
 * Note: We realized that sbb 24, 25, and 26 are not ori parts, but 5'UTRs for the rep parts. So we only proceeded with sbb 31 and 32.

Gel

 * 1) 31.1
 * 2) 31.2
 * 3) 32.1
 * 4) 32.2
 * Note: Decided to use 31.2 and 32.2

Project Change

 * Co-transformation of all Ori/5'UTR-Rep combinations
 * Check for colonies

Transformation
Transformation of ori: CA42, O99, P9 with rep: CA42, O99, P9 in all combinations [Ori/Rep] Plate 1: pir+,Spec/Kan CA42/CA42	CA42/O99	CA42/P9	O99/CA42	O99/O99	O99/P9	P9/CA42	P9/O99	P9/P9 pir-,Spec/Kan CA42/CA42	CA42/O99	CA42/P9	O99/CA42	O99/O99	O99/P9	P9/CA42	P9/O99	P9/P9 Plate 2: pir-,Spec CA42/CA42	CA42/O99	CA42/P9	O99/CA42	O99/O99	O99/P9	P9/CA42	P9/O99	P9/P9 pir-,Kan CA42/none	O99/none	 P9/none Transformation/Rescue/Plate
 * positive control
 * experimental
 * transformation control
 * negative control

Colony Inspection

 * CA42/CA42, CA42/P9, and P9/P9 had colonies
 * CA42/P9 might have also had colonies
 * Note: We will redo transformation.

Transformation
Transformation of ori: CA42, O99, P9 with rep: CA42, O99, P9 in all combinations [Ori/Rep] Plate 1: pir+,Spec/Kan CA42/CA42	CA42/O99	CA42/P9	O99/CA42	O99/O99	O99/P9	P9/CA42	P9/O99	P9/P9 pir-,Spec/Kan CA42/CA42	CA42/O99	CA42/P9	O99/CA42	O99/O99	O99/P9	P9/CA42	P9/O99	P9/P9 Plate 2: pir-,Spec CA42/CA42	CA42/O99	CA42/P9	O99/CA42	O99/O99	O99/P9	P9/CA42	P9/O99	P9/P9 pir-,Kan CA42/none	O99/none	 P9/none Transformation/Rescue/Plate
 * positive control
 * experimental
 * transformation control
 * negative control

Colony Inspection
Plate 1 Plate 2
 * CA42/CA42, CA42/P9, O99/P9, and P9/P9 had colonies

From First (4/26/2010) Transformation
Ori CA42 O99   P9          CA42  +    -    - Rep O99  -    -    - P9 +    ?    +

From Second (4/29/2010) Transformation
Ori CA42 O99   P9          CA42  +    -    - Rep O99  -    -    - P9 +    +    +