Part:BBa_K1403000

GFP generator in pSB1C3

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

Usage and Biology

This construct produce GFP at high levels as it is placed in a high copy plasmid and the J23101 Anderson's promoter is a strong promoter. </br>

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] sequenced used this part in the Interlab study to measure the expression level of the promoter comparing to the same promoter placed in low copy plasmid backbone (pSB1K3): comparing BBa_K1403000 and BBa_I20260. We also used this part to compare two different promoters BBa_K823012 (a version of BBa_J23101).

We observed that there is a variation depending on the plasmid and on the promoter.

We also sequenced the device.

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 which coresponds to pSB1C3 and another one a little bit below the 1kbp which coressponds 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 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 was determined from LB control.
Data from the top row were excluded due to the likely evaporation and artefacts (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) subtracted from the samples. Values 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 fluorescence (LB) subtracted from the samples. Values 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) subtracted from the samples. Values 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 [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 extract BBa_E0240 with 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. Quantified DNA in two parts with nanodrop. The amount of vector: insert has been calculated with 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
16°C overnight

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