Difference between revisions of "Part:BBa K535007"
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==Characterization==
 
==Characterization==
  
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===Segregational Plasmid Stability===
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===1. Segregational Plasmid Stability===
  
 
To determine the segregational stability of this plasmid we performed a technique called parallel plating of samples.
 
To determine the segregational stability of this plasmid we performed a technique called parallel plating of samples.
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* The Total count is given by the number of colonies in AB-free mediuim, i.e. the positive control
 
* The Total count is given by the number of colonies in AB-free mediuim, i.e. the positive control
  
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'''Legend:'''
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'''Legends:'''
 
* t -> Generations
 
* t -> Generations
 
* x -> Event: plasmid kept
 
* x -> Event: plasmid kept
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'''P(!x) = 0.000226'''  
 
'''P(!x) = 0.000226'''  
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===2. Plasmid copy number approximation===
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In order to measure the plasmid copy number we performed a comparative and qualitative method.
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We growth two ''E. coli'' DH5α populations that differed in the plasmid that they were carrying, one was carrying the plasmid pSB1T3 with an RFP inserted (part [https://parts.igem.org/Part:BBa_J04450 BBa_J04450]) and the other was carrying the plasmid pBBR1MCS-5 with the same RFP part.
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Knowing that these plasmids have different replication systems, we compared the RFUs (Relative fluorescence units) normalized to the optic density in which the RFUs were measured, during a period of 4 hours 20 minutes. During this time lapse, 13 measurements were taken every 20 minutes. The RFUs increased constantly in both populations, however we observed a 3.96:1 ratio in the RFU from the ''E. coli'' cells carrying the plasmid pSB1T3 compared to the ''E. coli'' cells carrying the pBBR1MCS-5 plasmid with the same RFP part.
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The replication system is the only variant between the two populations. Since the  replication system is directly involved in the number of plasmid copies in the cell, we conclude that the ratio seen in the RFUs is caused by the plasmid copy number. The ratio observed indicates that the plasmid we standardized is present in approximately four times less copies than the pSB1T3 plasmid.
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[[Image:Grafica_plasmidos.png|thumb|550px|center|Comparisons between the nomalized RFUs in a 4 hour, 13 messurments experiment. The RFUs were normalized by the optical density at which the measurement was taken. the time unit is 20 minutes.]]
  
  

Latest revision as of 03:54, 29 September 2011

pBBR1MCS-5 -> broad-host-range cloning vector

The plasmid we designed is a modification of the pBBR1MCS-5 , this broad-host-range (bhr) vector has been designed to assist the genetic analysis in prokaryotes, specifically in Gram-negative bacteria that are naturally CmR (chloramphenicol resistant).

The pBBR1MCS-5 vector is relatively small (4768bp), it has an extended multiple cloning site (MCS) with 15 different restriction sites and it possesses a gentamicin resistance gene. After modification, only 3 of the 15 retrictions sites are present (check out the Design Notes).

The plasmid is mobilizable when the RK2 transfer functions is provided in trans and is compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

We used this plasmid to transfer genes from E. coli to R. etli. First we transformed E. coli S17 with this vector containing the genes or sequences that we wanted to transfer, then we did a conjugation between our transformed E. coli cells and Rhizobium etli CFN42


Characterization

1. Segregational Plasmid Stability

To determine the segregational stability of this plasmid we performed a technique called parallel plating of samples.

This technique consists in plating same amounts of diluted samples on selective and non selective plates. In this case the selective plate consisted in a solid LB medium plate with 0.1% concentration of Gentamicin because pBBR1MCS-5 has a selection marker for this antibiotic.

The plasmid stability is represented by the ratio of colonies on the plates.

Experimental data

We performed an assay every 12 hour during 72 hours.

Hour(s) Colonies in non-selective medium Colonies in selective medium Ratio
0 100 100 1
12 100 100 1
24 97 96 0.9897
36 68 65 0.9559
48 100 98 0.98
60 100 99 0.99
72 100 99 0.99

Model data

In order to extrapolate the experimental results we generate a Markov model.

Assumptions:

  • The initial inocutalion at t=0 is of first daughter cells, i.e. second generation
  • Doubling time is of 40 minutes, conservative estimate for M9 glucose medium
  • There are no Gain-Of-Function mutations that generate phenotipe without plasmid
  • The Total count is given by the number of colonies in AB-free mediuim, i.e. the positive control

Legends:

  • t -> Generations
  • x -> Event: plasmid kept
  •  !x -> Event: plasmid Not kept
  • O(x)t -> Observance of x at time t
  • P(x)t -> Compound probability of x at time t since t=0
  • P(x) -> Probability of x for time t since time t-1


t Total O(x)t O(!x)t P(x)t P(x)
1 100 100 0 1 1
19 100 100 0 1 1
37 97 96 1 0.9897 0.9997
55 68 65 3 0.9559 0.9992
73 100 98 2 0.98 0.9997
91 100 99 1 0.99 0.9999
109 100 99 1 0.99 0.9999

P(x) = 0.999774

P(!x) = 0.000226


2. Plasmid copy number approximation

In order to measure the plasmid copy number we performed a comparative and qualitative method.

We growth two E. coli DH5α populations that differed in the plasmid that they were carrying, one was carrying the plasmid pSB1T3 with an RFP inserted (part BBa_J04450) and the other was carrying the plasmid pBBR1MCS-5 with the same RFP part.

Knowing that these plasmids have different replication systems, we compared the RFUs (Relative fluorescence units) normalized to the optic density in which the RFUs were measured, during a period of 4 hours 20 minutes. During this time lapse, 13 measurements were taken every 20 minutes. The RFUs increased constantly in both populations, however we observed a 3.96:1 ratio in the RFU from the E. coli cells carrying the plasmid pSB1T3 compared to the E. coli cells carrying the pBBR1MCS-5 plasmid with the same RFP part.

The replication system is the only variant between the two populations. Since the replication system is directly involved in the number of plasmid copies in the cell, we conclude that the ratio seen in the RFUs is caused by the plasmid copy number. The ratio observed indicates that the plasmid we standardized is present in approximately four times less copies than the pSB1T3 plasmid.

Comparisons between the nomalized RFUs in a 4 hour, 13 messurments experiment. The RFUs were normalized by the optical density at which the measurement was taken. the time unit is 20 minutes.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Illegal EcoRI site found at 5006
    Illegal XbaI site found at 5021
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5006
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 3127
    Illegal NotI site found at 5012
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5006
    Illegal BglII site found at 3927
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Illegal suffix found at 2
    Illegal EcoRI site found at 5006
    Illegal XbaI site found at 5021
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5006
    Illegal XbaI site found at 5021
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 855
    Illegal AgeI site found at 695
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 3449