Part:BBa_K535007
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
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 several assays 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
Legend:
- 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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Plasmid lacks a prefix.
Illegal EcoRI site found at 5006
Illegal XbaI site found at 5021 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5006
Illegal BglII site found at 3927 - 23INCOMPATIBLE WITH RFC[23]Plasmid lacks a prefix.
Illegal suffix found at 2
Illegal EcoRI site found at 5006
Illegal XbaI site found at 5021 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI.rc site found at 3449
| None |