iGEM 2014 Measurement Interlab

1. First we transformed E. coli Dh5alpha according to iGEM protocol with the following parts from iGEM distribution kit 2014:
• BBa_I20260 (Plate 4, Well 18A)
• BBa_J23101 (called BBa_K823005 when in pSB1C3): Plate 1, Well 20K
• BBa_E0240 (in pSB1C3): Plate 2, Well 24B
• BBa_J23115 (called BBa_K823012 when in pSB1C3): Plate 1, Well 22I
• BBa_E0240 (in pSB1C3): Plate 2, Well 24B

2. After we observed the growth of transformed bacteria on chloramphenicol selective plates, each part was extracted using PureLink® HiPure Plasmid Miniprep Kit (Invitrogen, catalog number K2100-02). The concentration of the plasmids was checked with Nanodrop.

PCR of the parts was done according to the following protocol for 20ul reaction (adapted from http://www.thermoscientificbio.com/uploadedFiles/Resources/tech-manual-f-531-f-532-phusion-high-fidelity-pcr-master-mix.pdf):
• Sterile water – 4ul, high fidelity master mix – 10ul, VF2 primer – 2ul, VR primer – 2ul, DNA from miniprep (final concentration in the reaction=0.2ng/ul) – 2ul.
• Thermocycler program for the parts was adapted from http://www.thermoscientificbio.com/uploadedFiles/Resources/tech-manual-f-531-f-532-phusion-high-fidelity-pcr-master-mix.pdf and the extension time was adjusted according to the size of the part (25s per 1000bp).


Figure 1. Agarose gel electrophoresis of PCR products: first lane – 500bp gene ladder, second and third lanes – PCR products of Anderson promoters for Interlab study (35bp each)



Figure 2. Agarose gel electrophoresis of PCR product of GFP generator for Interlab study: 1st lane – 1kb gene ruler, 2nd lane – 876bp GFP generator amplified with PCR.


Part construction
We used protocols provided for endonucleases from Invitrogen and Fermentas for restriction digest. Furthermore, we used circular polymerase extension cloning (CPEC).
• Protocol for restriction digest of BBa_J23101 and BBa_J23115 (separately): DNA (PCR product) – 10ul, sterile water – 17ul, 10xTango buffer – 2ul, SpeI (Fermentas) – 1ul. Incubate the reactions for 2 hours at 370C.
• Protocol for restriction digest of BBa_E0240:
DNA from pcr – 8ul, sterile water – 17ul, 10xM buffer – 2ul, 0.1% BSA – 2ul, XbaI (Invitrogen) – 1ul. Incubate the reaction for 2 hours at 370C.
• Protocol for CPEC for inserting promoter and GFP into backbone for one 25ul reaction (Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. doi:10.1371/journal.pone.0006441)
Reaction mixture:
• Linearized plasmid backbone – 4ul
• Promoter digested with SpeI – 2ul
• BBa_E0240 digested with XbaI – 2ul
• High Fidelity master mix (Invitrogen) – 11.75ul
• DMSO – 0.75ul
• Sterile water – 4.5ul

Construct 1: BBa_J23101+ BBa_E0240 (size of the part together with the PSB1C3 backbone is 2981: 2070bp+35bp+876bp)

Construct 2: BBa_J23115 +BBa_E0240 (size of the part together with the PSB1C3 backbone is 2981: 2070bp+35bp+876bp)

Results: We observed the bands of the expected sizes on the gel, which means that the desired incorporation did occur (Figure 3).

Figure 3. Agarose gel electrophoresis of CPEC products: 1st lane – 1kb gene ruler, 2nd and 5th lanes – Promoter (BBa_J23101)+INP construct (BBa_I20260), 3d and 6th lanes – Promoter (BBa_J23101)+GFP generator (BBa_E0240), 4th and 6th lanes – Promoter (BBa_J23115)+GFP generator (BBa_E0240)

To check the expression of the constructed parts we transformed E. coli Dh5alpha with CPEC products and grew the bacteria on LB agar plates with chloramphenicol. After the growth of the colonies was observed, a single colony of bacteria was inoculated into 5ml of LB medium with the appropriate antibiotic concentration and IPTG:
1. Bacteria with BBa_I20260 into 5ml LB, 1mM IPTG and 30ug/ml Kanamycin
2. Bacteria with Construct 1 into 5ml LB, 1mM IPTG and 35ug/ml Chloramphenicol
3. Bacteria with Construct 2 into 5ml LB, 1mM IPTG and 35ug/ml Chloramphenicol
4. Control (Bacteria with BBa_J45014) into 5ml LB, 1mM IPTG and 35ug/ml Chloramphenicol

The cultures were incubated for 20 hours at 200rpm at 370C. The cultures were put into 96-well Corning, flat bottom plate were used and GFP intensity was measured with Thermo Scientific Varioskan® Flash

Table 1. Layout of 96-well Corning, flat bottom plate. 18a represents the existing device BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector from Plate 4, Well 18A; 200, 175, and 150 are volume of liquid cultures in each well in microliters (175ul were mixed with 25ul of LB medium, 150ul – with 50ul of LB); blank is LB medium.

Photometric and fluorometric measurements were recorded and analyzed.

The measurements were taken on Varioskan according to the protocol below:

After the measurements were taken, the analysis of the data obtained was done.

Figure 4. Photometric intensity (RFU, relative fluorescence units) of GFP under different promoters; measured at different wavelengths

Figure 5. Fluorometric measurement of GFP intensity (RFU) under different promoters; the error bars represent standard deviation

Table 2. The mean and standard deviation values for each quantity measured. Replicate 1: 150ul of culture+50ul of media, Replicate 2: 175ul of culture+25ul of media, Replicate 3: no dilution