Part:BBa_K1403000

GFP generator in pSB1C3

Measurment kit test of Part:BBa_J23101 in pSB1C3. This part is a tween of BBa_I20260 in a high copy pSB1C3 plasmid.

Usage and Biology

This construct produces GFP at high levels as it is placed in a high copy plasmid under the J23101 Anderson's strong promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 706
  

Applications of BBa_K1403000

Interlab Study
[http://2014.igem.org/Team:Paris_Bettencourt# iGEM Paris Bettencourt 2014] used BBa_K1403000 in an interlab study to measure the expression level of the promoter compared to the expression of that promoter when placed in low copy plasmid backbone (pSB1K3),BBa_I20260. We also assessed the impact on GFP expression when the promoter is changed for a given plasmid by comparing BBa_K1403000 to BBa_K823012 (a version of BBa_J23101). We observed variations in expression dependent on both the plasmid and the promoter.

We also sequenced each device.

The difference between the constructs and intensity could be observed by the naked eye through the filter.
Device 1: |BBa_I20260
Device 2: |BBa_K1403000
Device 3: |BBa_K1403001

Verification

We cut the construct with the EcoRI and PstI enzymes in order to verify it. Gel was run with the 1kb plus ladder.

We obtained two bands. One around 2kbp corresponding to pSB1C3 and another one a little bit below the 1kbp, which corresponds to 960 bp for promoter + RBS+ GFP + terminators (Part:BBa_J23101 + BBa_E0240(B0032-E0040-B0015)). This result confirms again that the construct follows the design.

Measurment

Samples preparation
Single colonies were inoculated in 5mL LB broth with the appropriate antibiotic and grown to saturation overnight (16h) at 37°C with shaking (220 rpm). Samples were diluted 100x (50um in 5 mL LB with appropriate antibiotic) and incubated for 2h at 37°C prior to measurement.

Control
LB broth with antibiotics (chloramphenicol/kanamycin) - no fluorescence.
NEB turbo without fluorescence - no fluorescence, no cells.

Measurment
Greiner 96 plates were loaded with 150um of cells in LB and 30um mineral oil. Cells have been diluted prior to measurement as described above. Background absorbance and fluorescence were determined from LB control.
Data from the top row were excluded due to the high likelihood of evaporation and artifacts (edge effects).

Device 1: |BBa_I20260
Device 2: |BBa_K1403000
Device 3: |BBa_K1403001

Fig.1. Mean OD600 absorbance measured over 20h. Background absorbance (LB) has been subtracted from the samples. Values are reported in the log scale with error bars for standard deviation (7 replicates for each sample).

Fig.2. Mean of green fluorescence for three devices and NEB turbo cells. Background has been fluorescence (LB) subtracted from the samples. Values are reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).

Fig.3. Mean of green fluorescence divided by optical density 600 for three devices and NEB turbo cells. Background fluorescence (LB) has been subtracted from the samples. Values are reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).

Part construction protocol

We followed iGEM Distribution Kit instructions to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then for the [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1# Heat Shock transformation of E.coli]. For successful Chloramphenicol plates, form single colonies we prepared liquid cultures overnight. We used 750uL of the liquid cultures for a [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8# glycerol stock] . We used remaining 4,25 mL to make [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot4# minipreps]. We measured DNA content with the nanodrop.

Digestion analysis:
- 5 ug plasmid
- 5 ul FD Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)

We made an eletrophoresis gel to check the fragments (the bands at around 876 bp for GFP and 2100 bp for the promoter + backbone) and then extracted BBa_E0240 with a gel extraction kit. For the plasmid with the promoter we used a [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot5# PCR purification kit]. We introduced the GFP fragment to the promoter + backbone through ligation of the sticky ends SpeI and XbeI. DNA was quantified in two parts with nanodrop. The amount of vector insert has been calculated with the Promega calculator.

5X Ligase Reaction Buffer: 4 μl
Insert: Vector Molar Ratio 1:1, 1:3, 1:5
Total DNA: 0.01-0.1 μg
T4 DNA Ligase: 1 uL
Autoclaved distilled water to 25uL
Incubate at 22°C for 1h
Incubate at 16°C overnight

We transformed the ligation product following the [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot1# heat shock transformation]. We put a single colony into a liquid culture with the appropriate antibiotic and prepared a [http://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8# glycerol stock] the next day.