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*Andrew Saarni<br> | *Andrew Saarni<br> | ||
*[[User:Kailin Mesa|Kai Mesa]]<br> | *[[User:Kailin Mesa|Kai Mesa]]<br> | ||
*Jose Gutierrez<br> | *[[User:Jose Gutierrez|Jose Gutierrez]]<br> | ||
*Zhen Huang<br> | *Zhen Huang<br> | ||
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#Transform Rep plasmid into Bacteria (Ec100D) | #Transform Rep plasmid into Bacteria (Ec100D) | ||
#Plate on ( | #Plate on (Kan) plates, Pick Colonies | ||
#Miniprep for plasmid DNA purification | #Miniprep for plasmid DNA purification | ||
Line 131: | Line 131: | ||
#Once first transformation is done, make competent cells using colony | #Once first transformation is done, make competent cells using colony | ||
#Repeat fist four steps with Ori Plasmid, Plate on ( | #Repeat fist four steps with Ori Plasmid, Plate on (Kan/Spec) plates | ||
Line 246: | Line 246: | ||
====Digest==== | ====Digest==== | ||
*[[Template:SBB-Protocols Enz2|Digest]] | *[[Template:SBB-Protocols Enz2|Digest]] | ||
**Note: We realized that sbb 24, 25, and 26 are not ori parts, but 5'UTRs for the rep parts.<br> | **Note: We realized that sbb 24, 25, and 26 are not ori parts, but 5'UTRs for the rep parts.<br> So we only proceeded with sbb 31 and 32. | ||
====Gel==== | ====Gel==== | ||
Line 254: | Line 254: | ||
# 32.1 | # 32.1 | ||
# 32.2 | # 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 | |||
==4/26/2010== | |||
===Transformation=== | |||
'''Transformation of ori: CA42, O99, P9 with rep: CA42, O99, P9 in all combinations''' | |||
<br> | |||
['''Ori'''/'''Rep'''] | |||
<br> | |||
'''Plate 1:''' <br> | |||
pir+,Spec/Kan | |||
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9 | |||
*positive control | |||
pir-,Spec/Kan | |||
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9 | |||
*experimental | |||
'''Plate 2:''' <br> | |||
pir-,Spec | |||
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9 | |||
*transformation control | |||
pir-,Kan | |||
CA42/none O99/none P9/none | |||
*negative control<br> | |||
[[Template:SBB-Protocols Micro1|Transformation/Rescue/Plate]] | |||
==4/27/2010== | |||
===Colony Inspection=== | |||
*CA42/CA42, CA42/P9, and P9/P9 had colonies | |||
*CA42/P9 might have also had colonies | |||
**Note: We will redo transformation. | |||
==4/29/2010== | |||
===Transformation=== | |||
'''Transformation of ori: CA42, O99, P9 with rep: CA42, O99, P9 in all combinations''' | |||
<br> | |||
['''Ori'''/'''Rep'''] | |||
<br> | |||
'''Plate 1:''' <br> | |||
pir+,Spec/Kan | |||
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9 | |||
*positive control | |||
pir-,Spec/Kan | |||
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9 | |||
*experimental | |||
'''Plate 2:''' <br> | |||
pir-,Spec | |||
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9 | |||
*transformation control | |||
pir-,Kan | |||
CA42/none O99/none P9/none | |||
*negative control<br> | |||
[[Template:SBB-Protocols Micro1|Transformation/Rescue/Plate]] | |||
==4/30/2010== | |||
===Colony Inspection=== | |||
[[Image:Picture 3.png]]'''Plate 1''' | |||
[[Image:Picture 2.png]]'''Plate 2''' | |||
*CA42/CA42, CA42/P9, O99/P9, and P9/P9 had colonies | |||
==Results== | |||
===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 '''+''' '''+''' '''+''' |
Latest revision as of 15:54, 3 May 2010
Team 3 Info
Members
- Andrew Saarni
- Kai Mesa
- Jose Gutierrez
- Zhen Huang
Assay Guidelines
3/31/2010
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.
4/5/2010
Second Group Meeting
Worked on Project Presentation
Team 3
[ColE2 Ori / Rep System]
-Origin Stability and Copy Number-
---
[Objective]
-Design assays to...
- Test the orthogonality and stability of regulation for 27 different ColE2 replicons, prepared by combining different Ori/Rep plasmids and promoter regions
- 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
- Transform Rep plasmid into Bacteria (Ec100D)
- Plate on (Kan) plates, Pick Colonies
- Miniprep for plasmid DNA purification
- Analytical digest
- Once first transformation is done, make competent cells using colony
- 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.
- need a way to standardize relative # of Ori plasmids/cell
- Rep plasid copy should be constant for each cell
- isolate both plasmids, and map
- 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
4/7/2010
Project Presentations
4/12/2010
Eco/Bam Transfer
Ori basic parts need to be transfered into R6K vectors
- We perfromed an Eco/Bam on parts: sbb24, sbb25, sbb26, sbb31 and sbb32 as well as an R6K vector.
- Set up the digest:
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
- Incubate for 1hr at 37°C
- Zymo Cleanup
- We used 10μl of ddH2O for the elution step in Zymo cleanup.
4/14/2010
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.
- Set up the digest:
7uL ddH2O 2uL NEB2 Buffer 1uL EcoRI 1uL BamHI 9uL R6K plasmid
- Incubate for 1hr at 37°C
- Run out on gel
- 1400bp band is our R6K vector
- Cut out band and purify
4/19/10
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.
4/20/2010
Colony Inoculation
- Colony Picking
- Note: 2 colonies picked per plate
4/21/2010
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)
Mapping
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.
- Note: We realized that sbb 24, 25, and 26 are not ori parts, but 5'UTRs for the rep parts.
Gel
- 31.1
- 31.2
- 32.1
- 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
4/26/2010
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
- positive control
pir-,Spec/Kan CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9
- experimental
Plate 2:
pir-,Spec
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9
- transformation control
pir-,Kan CA42/none O99/none P9/none
- negative control
4/27/2010
Colony Inspection
- CA42/CA42, CA42/P9, and P9/P9 had colonies
- CA42/P9 might have also had colonies
- Note: We will redo transformation.
4/29/2010
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
- positive control
pir-,Spec/Kan CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9
- experimental
Plate 2:
pir-,Spec
CA42/CA42 CA42/O99 CA42/P9 O99/CA42 O99/O99 O99/P9 P9/CA42 P9/O99 P9/P9
- transformation control
pir-,Kan CA42/none O99/none P9/none
- negative control
4/30/2010
Colony Inspection
- CA42/CA42, CA42/P9, O99/P9, and P9/P9 had colonies
Results
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 + + +