Team:TrinityCollegeDublin/Measurement
Interlab Study
As 2015 is the first year that a team from Trinity College Dublin has participated in the iGEM competition, we have decided to involve ourselves in one of the side projects that helps unite numerous teams every year; the Interlab Study. Its objective is to obtain fluorescence data for three constructs i.e. Biobrick devices expressing GFP, chosen by iGEM Headquarters. Comparison of data sent by teams from all over the world serves as a means to evaluate its consistency between different labs. Devices were assembled from DNA parts available from the Distribution Kit 2015. Those devices differ in GFP promoter strength, which can be visualised by measuring their fluorescence.
We have found the Interlab study to be a valuable experience which allowed us, 2nd year Human Genetics students, to master many basic techniques, such as making competent cells, bacterial transformations, miniprepping and various other skills, which are crucial for us as future geneticists.
We are excited to share our data with other iGEM Teams 2015 as well as any subsequent teams willing to follow in our footsteps!
J23101 (called K823005 when in pSB1C3 backbone), J23106 (K823008) and J23117 (K823013) are promoters belonging to the same family, with different expression strength. J23101 and J23117 are the strongest and weakest promoters tested, respectively. All were available from Distribution Kits 2015 and are built in pSB1C3 backbones, carrying chloramphenicol resistance. I13504 is a high copy plasmid with GFP generator. It is built in pSB1A2 backbone carrying ampicillin resistance.
XL1-Blue E.coli competent cells were prepared and transformed with the assembled plasmids. Once assembled, 3 broth cultures per device (for the biological triplicates) were prepared and incubated overnight for 18 hours and then taken to the flow cytometry facility to measure the respective fluorescence of each device in biological as well as technical replicates.
Since the promoters were already attached to PSB1C3 backbones in the iGEM kits, we figured it was unnecessary to use a separate linearized plasmid backbone, as we had done previously. The main problem lied in the backbone circularizing itself without taking in the GFP insert. Therefore, a 2A assembly protocol was followed instead, with part A being the promoter with the backbone, and part B being the GFP insert. The plasmids were recovered using the MiniPrep Spin Kit provided by iGEM, and their concentrations were tested using a nanodrop.
The purified plasmids (promoter with backbone) were cut with the restriction enzymes EcoRI and SpeI, while the GFP insert was cut with XbaI and PstI. The restricted plasmids were then ligated together using the T4 DNA Ligase. All restrictions and ligations were performed using enzymes and buffers of the 2015 iGEM Kit (with the exception of using buffer 2.1 from the genetics lab for restriction protocol). The final product was once again transformed into Xl1-blue cells using the iGEM transformation protocol. The correct identity of the resulting constructs was confirmed by running a gel i.e. lanes corresponding to devices 1-3 and the positive control have bands on the same level, proving the presence of GFP insert that the negative control is lacking.
Each measurement occurred in a technical triplicate, resulting in 9 samples being processed for every device. The results were analysed using Microsoft Excel 2013.
The ratios of the promoter strength, however, aren’t exactly matching the ones given on the iGEM page. The strongest promoter J23101 was much stronger than was expected, being 4.7 times stronger than Device 2 and 96 times stronger than Device 3. J23106 is approximately 20 times stronger than J23117.
We have found the Interlab study to be a valuable experience which allowed us, 2nd year Human Genetics students, to master many basic techniques, such as making competent cells, bacterial transformations, miniprepping and various other skills, which are crucial for us as future geneticists.
We are excited to share our data with other iGEM Teams 2015 as well as any subsequent teams willing to follow in our footsteps!
Experimental design
The three devices to be measured included:- Device 1: J23101 + I13504 (B0034-E0040-B0015) built in pSB1C3 backbone
- Device 2: J23106 + I13504 (B0034-E0040-B0015) built in pSB1C3 backbone
- Device 3: J23117 + I13504 (B0034-E0040-B0015) built in pSB1C3 backbone
J23101 (called K823005 when in pSB1C3 backbone), J23106 (K823008) and J23117 (K823013) are promoters belonging to the same family, with different expression strength. J23101 and J23117 are the strongest and weakest promoters tested, respectively. All were available from Distribution Kits 2015 and are built in pSB1C3 backbones, carrying chloramphenicol resistance. I13504 is a high copy plasmid with GFP generator. It is built in pSB1A2 backbone carrying ampicillin resistance.
Controls
In addition, we have also obtained measurements of negative and positive controls, as follows:- Positive control: BBa_I20270, a GFP expression device in the pSB1C3 backbone.
- Negative control #1: empty cells with no plasmid added.
- Negative control #2: competent cells transformed with an empty vector BBa_R0040, which is pTetR in pSB1C3.
XL1-Blue E.coli competent cells were prepared and transformed with the assembled plasmids. Once assembled, 3 broth cultures per device (for the biological triplicates) were prepared and incubated overnight for 18 hours and then taken to the flow cytometry facility to measure the respective fluorescence of each device in biological as well as technical replicates.
Expected Results:
All cultures apart from the negative controls were expected to show fluorescence. The signal was expected to be the strongest in Device 1 (the one with the J23101 Anderson promoter) followed by Device 2, and Device 3 was expected to show the weakest signal. The theoretical ratios for the promoter strength obtained from the iGEM website, (parts.igem.org/Promoters/Catalog/Anderson), were 1.00:0.67:0.08 for Devices 1, 2 and 3, respectively.Materials and Methods
Initially, we used biobrick assembly method (3A assembly protocol) to construct the devices. However, it didn’t prove most efficient, as we only obtained one right colony for Device 2, and none for devices 1 and 3. Thus, after restreaking the one colony from Device 2, an amended method was adopted to reconstruct Devices 1 and 3.Since the promoters were already attached to PSB1C3 backbones in the iGEM kits, we figured it was unnecessary to use a separate linearized plasmid backbone, as we had done previously. The main problem lied in the backbone circularizing itself without taking in the GFP insert. Therefore, a 2A assembly protocol was followed instead, with part A being the promoter with the backbone, and part B being the GFP insert. The plasmids were recovered using the MiniPrep Spin Kit provided by iGEM, and their concentrations were tested using a nanodrop.
The purified plasmids (promoter with backbone) were cut with the restriction enzymes EcoRI and SpeI, while the GFP insert was cut with XbaI and PstI. The restricted plasmids were then ligated together using the T4 DNA Ligase. All restrictions and ligations were performed using enzymes and buffers of the 2015 iGEM Kit (with the exception of using buffer 2.1 from the genetics lab for restriction protocol). The final product was once again transformed into Xl1-blue cells using the iGEM transformation protocol. The correct identity of the resulting constructs was confirmed by running a gel i.e. lanes corresponding to devices 1-3 and the positive control have bands on the same level, proving the presence of GFP insert that the negative control is lacking.
Inoculation and Cultivation
Broth cultures of the devices and controls were prepared in sterile conditions. Each construct had three assigned falcon tubes containing broth made of 5 mL LB medium with 5 µL Chloramphenicol antibiotic (from stock solution of 25 mg/ml). Three separate colonies from each Device plate were picked using a pipette tip and transferred to the respective falcon tubes. For the negative control containing bacterial cells only, tetracycline was used as the antibiotic, as Xl1-Blue cells are tetracycline resistant. The cultures were kept at 37°C and 300 rpm shaking frequency upright for 18 hours overnight.Sampling
To draw samples, the tubes were taken out of the 37°C shaker and brought into the flow cytometry facility. 1 ml of sample from each of the 18 tubes was taken out next to a Bunsen burner flame and pipetted into 2 ml clean cuvettes, which were then vortexed and fluorescence was measured from median values using FACS (fluorescence-activated cell sorting) to compare the different promoter strengths in relative arbitrary units.Each measurement occurred in a technical triplicate, resulting in 9 samples being processed for every device. The results were analysed using Microsoft Excel 2013.
Flow Cytometry details
The BD Accuri C6 Flow Cytometer (a 2 laser, 4 colour instrument) was calibrated right before the experiment with the use of sphere beads (8Pk) by Mr.Barry at 488 nm (530/30 peak-range).Results
Discussion
As can be seen from Table 1 and Figure 2, the data tallies to the expected results: almost negligible fluorescence is detected in negative controls with the empty vector and the Xl1-blue cells, whereas cells transformed with I20270 (positive control) show intermediate fluorescence. Device 1 has the strongest fluorescence, followed by device 2, and finally device 3 which has the weakest promoter strength.The ratios of the promoter strength, however, aren’t exactly matching the ones given on the iGEM page. The strongest promoter J23101 was much stronger than was expected, being 4.7 times stronger than Device 2 and 96 times stronger than Device 3. J23106 is approximately 20 times stronger than J23117.
Other data
- Marlena Mucha and Remsha Afzal carried out the preparation of the competent cells and testing for their transformation efficiency.
- The biobrick assembly method (restriction/ligation etc.) was done by Marlena Mucha, Arnas Petrauskas and Remsha Afzal.
- Remsha Afzal and Arnas Petrauskas carried out the final transformations of the constructed devices into competent Xl1-Blue cells and made the broth cultures for measurement.
- Barry Moran from the flow cytometry facility showed us how to use the FACS machine and its software and helped us analyse the readings.
- Data was processed by Remsha Afzal.
- The measurements for the InterLab study using FACS took place on the 20th of July 2015.