Biobrick
CONTENTS:
List of Biobricks:
BBa_K094100, BBa_K094101, BBa_K094102, BBa_K094103, BBa_K094104, BBa_K094105, BBa_K094106, BBa_K094110, BBa_K094111, BBa_K094112, BBa_K094113, BBa_K094120, BBa_K094130, BBa_K094141, BBa_K094150, BBa_K094151
Characterization of BBa_pSB2K3
List of Biobricks:
BBa_K094100 
cheZ: This part contains the cheZ operon coding region without an rbs nor terminator. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement.
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BBa_K094101 
plambda(R-O12)-rbs-cheZ: This part contains a lambda promoter that is constitutively on and can be repressed by CI protein. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement. This part lacks a terminator.
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BBa_K094102 
pTetR-rbs-cheZ: This part uses BBa_J61003 as a backbone and cheZ as the inserted gene. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement. The cheZ operon lacks a terminator.
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BBa_K094103 
plux-rbs-cheZ-terminator: This is a cheZ gene that can be induced by luxR protein under high AHL concentration. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement.
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BBa_K094104 
pLacI/ara-1-rbs-mRFP-double terminator: This part is adapted from part BBa_J04450 by changing the promoter pLacI to promoter pLacI/ara-1. This part can test the leaky of promoter plac/ara-1.
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BBa_K094105 
plambda(R-O12)-rbs-cheZ:-rbs-mRFP1-double terminator: This part can be used to measure the gene expression of cheZ under the control of promoter plambda(R-O12).
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BBa_K094106 
Plux-rbs-CI-terminator-plambda-rbs-cheZ: This part encodes a CI protein that is used to repress lambda promoter. The part is controlled by the lux promoter that can be induced by luxR protein under high AHL concentration. In this part, the expression of cheZ is repressed by increasing AHL concentration in the environment in the presence of luxR protein. In other circumstances, the cheZ gene express constitutively.
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BBa_K094110 
cheY*: This is the gene coding for cheY protein which controls flagella movement. This sequence, however, is mutated to give a mutant cheY protein which will not respond to the presence of cheZ protein. This part contains the coding region only.
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BBa_K094111
pTetR-rbs-cheY*: This part uses BBa_J61003 as a backbone and cheY* as the inserted gene. cheY is the gene coding for cheY protein which controls flagella movement. This sequence, however, is mutated to give a mutant cheY protein which will not respond to the presence of cheZ protein. This part lacks a terminator.
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BBa_K094112 
luxR-rbs-luxI-terminator: : The bacterial luciferin-luciferase system is encoded by a set of genes labelled the Lux operon. In V. fischeri five such genes (LuxCDABE) have been identified as active in the emission of visible light, and two genes (LuxR and LuxI) are involved in regulating the operon. This part can be utilized as a part of quorum sensing system.
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BBa_K094113
lacZ: This is a reporter gene that codes for beta galactosidase.
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BBa_K094120 
pLacI/ara-1: This promoter can be repressed by lacI protein and induced by either IPTG or arabinose.
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BBa_K094130 
rbs-mRFP1-terminator: This part is adapted from part BBa_J04450, with the pLacI promoter deleted. This part can serve as promoter efficiency test kit.
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BBa_K094141 <
pLacIq-rbs-LacIq-terminator: LacIQ can repress promoter lac. This part contains a mutated pLacIq promoter which enables high level of transcription. This ensures effective repression of pLacI promoter that might be present.
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BBa_K094150 
pLacI/ara-1-rbs-luxI-luxR-terminator: This is a quorum sensing composition. Induced by IPTG or arabinose, this part can establish a quorum sensing system, with the luxI protein producing AHL and luxR protein as the AHL density sensor.
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BBa_K094151 
pLacI/ara-1-rbs-luxI-terminator: The luxI operon is controlled by promoter LacI/ara-1.
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Characterization: Effect of IPTG inducement on BBa_pSB2K3 copy number
Plasmid pSB2K3 is a variable copy number plasmid that carries one F’ replication origin and a P1 lytic replication origin. The P1 lytic origin must be activated by proteins encoded by a gene carried by the plasmid itself. The gene, however, is controlled by a pLacI promoter and is thus inducible by IPTG. When the plasmid is inserted into cells producing lacI protein, the P1 lytic origin is non-functional and its only active origin is the F’ origin that keeps plasmid copy number at a very low level, possibly one or two (which is determined by the nature of F’ origin). When induced by IPTG, the P1 lytic origin is activated and thus may keep copy number at a very high level.
To find out the relationship between the concentration of inducing IPTG and the copy number of the plasmid, we performed a series of experiments.
Method:
The common method for plasmid copy number determination is using real-time PCR. However, real-time PCR is not available for our experiment, so the method we used was plasmid extraction, gel-electrophoresis followed by brightness analysis.
Procedure:
1. Pre-culture of pSB2K3 containing cells in 2% LB medium with Kanamycin concentration of 50mg/L. (not induced with IPTG)
2. When O.D. has reached 1, begin the experimental culture with a 1:100 dilution of the pre-culture into 2% LB medium containing Kanamycin and IPTG. 10 different IPTG concentrations were selected and they are 0, 10, 20, 40, 60, 80, 100, 200, 500, 1000 (unit: μmol/L).
3. Wait until the experimental culture has reached an O.D. close to 1. Measure the O.D. and calculate the volume of sample to be collected to make it contain the same cell mass as 5ml of O.D.=1.000 culture. Then collect the 10 samples and put them on ice.
4. Centrifuge down the cells and extract plasmid using a QIAGEN extraction kit. The extraction is done with extreme carefulness and strict protocol to ensure a high recovery and high repeatability.
5. The extracted plasmid is run with 1.0% agarose gel. 1kb plus DNA ladder is used as a control of DNA amount and plasmid size. The plasmid is digested with XbaI before gel electrophoresis.
6. Photo of the gel result is analyzed by brightness measuring software.
Rationale and result interpretation:
The experiment is repeated twice (altogether three times) to ensure the repeatability of this method. The values shown in the result chart (figure 1) are the mean values of the three experiments with error bar. Figure 2 is an enlarged part of figure 1 showing the first two data.
Figure 1
Figure 2
Six data are shown in the chart, because for IPTG concentration larger than 100 μmol/L the effect of inducement seems to have no difference. Attached in the chart is one of the original gel photos, with C being the control. This data is got from exponentially growing culture, in which the mean copy number is generally smaller than the mean copy number of cells from stationary phase, for the simple reason that in newly divided cells there is not enough time for plasmids to replicate to the limit.
The copy number is calculated from the following statistics: cell number at O.D.=1 from our experimental result; plasmid molar weight calculated from mean nucleotide weight and the size of the plasmid; volume of cell culture sample and final volume of plasmid solution eluted (50 μl); the DNA amount in 1kb plus ladder (control) and the light intensity got from the gel photo by brightness analysis software. The calculation is very complicated and is omitted here.
To make sure that the brightness of DNA bands corresponds with the amount of DNA in it, control experiments with 1kb plus ladder of different amount to see if there amounts are proportional to their brightness intensity. The result is shown in figure 3 and we can see that the result is quite linear. The experiment is also repeated twice and the data is the mean value with error bar.
Figure 3
The result shows that for IPTG concentration less than 20 μmol/L, the effect of inducement is scarcely perceivable. It also shows that from 20 μmol/L to 100 μmol/L the copy number is induced by IPTG in a way quite proportional to its concentration. It seems that over 100 μmol/L IPTG concentration is “saturated” and a further increase in IPTG concentration cannot raise the copy number (the data of which is not shown in the chart).
Attention*: This result is from exponentially growing culture. For cell culture in stationary phase the copy number possibly increases (due to time limit, only one experiment is done with stationary phase culture and the result seems to suggest that this raise in plasmid copy number is more significant with lower IPTG concentration. Because there are no repeats, the data is not included).
According to the experiment, we suggest that 100 μmol/L IPTG is enough for pSB2K3 to reach its peak copy number, and if moderate copy number is needed then 40-60 μmol/L is a good choice. This result is also valuable when using pSB2K3 with other IPTG induced genetic devices.
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