The First InterLab Study in Synthetic Biology!
ABSTRACT
For interlab study, we started our work with examining the parts. We made a plan, so that we will start with transformation of the parts. However while doing our transformations we consumed all of the parts from 2014 kits. So we decided using twins of the parts from the previous kits.
First Device
The first device(BBa_I20260) was already constructed by iGEM. So we only did transformation for this part. We cultivated transformed cells to LB Agar plates with antibiotic resistance to make sure that the cell took the plasmid. After they grew in plates with resistance we took a single colony and inoculated it into 5 ml LB and waited in shaker incubator at 37 C 200 rpm for 16 hours which will be mentioned as overnight culture protocol. Then we used BD Accurium TM C6 Flow Cytometer for measurement. We used standard cytometer protocol done by our instructor. The first set of samples for the “existing device” took 24.7 seconds, 32.6 Seconds, 32.7 seconds. The results for the 3 samples of first device was like that First Device: BBa_I20260 in the pSB3K3 vector:
DV1-S1: All 1,194.19 (Mean Fluorescence Intensity)
DV1-S2: All 1,079.89 (Mean Fluorescence Intensity)
DV1-S3: All 1,165.72 (Mean Fluorescence Intensity)
(DV: Device, S: Sample)
-The first device has measured for three times: BBa_I20260 in the pSB3K3 vector
The Mean: 1,146.6
The Standard Deviation: 59.50045
Second Device
For the second device the parts was BBa_J23101 and BBa_E0240. We took BBa_J23101 from 2013 kit plate 5 well 18E. We set it as vector and digested it from E and X sites. BBa_E0240 as an insert was taken from 2013 kit plate 5 well 12 M and digested from E and P sites. Then we ligated them and put in one plasmid. This plasmid was later taken into a competent cell by transformation. Then we performed overnight culture protocol. At 16th hour of that we done measurements with flow cytometer. It gave the following results The second set which is “New Device (BBa_J23101+BBa_E0240)” took 2.1 Seconds, 2.0 Seconds, 2.1 Seconds.
C01 DV2-S1 : All 1,416.72 (Mean Fluorescence Intensity)
C02 DV2-S2 : All 1,371.77 (Mean Fluorescence Intensity)
C03 DV2-S3 : All 1,345.70 (Mean Fluorescence Intensity)
(DV: Device, S: Sample)
-The second device has measured for three times: BBa_J23101 + BBa_E0240 in pSB1C3 backbone
The Mean: 1,378.06
The Standard Deviation: 35.92582
Third Device
Third device was composed of BBa_J23115 from 2013 kit plate 5 well 20K as vector and BBa_E0240 from 2013 kit plate 5 well 12M as insert. We digested BBa_J23115 from X and P sites and BBa_E0240 from S and P sites. Then ligated them. The following procedures were same as the device 2 (transformation,overnight culture). After that we done measurements with cytometer and the results were
-Third Device: BBa_J23115 + BBa_E0240 in pSB1C3 backbone:
D01 DV3-S1 : All 1,419.34 (Mean Fluorescence Intensity)
D02 DV3-S2 : All 1,355.00 (Mean Fluorescence Intensity)
D03 DV3-S3 : All 1,123.69 (Mean Fluorescence Intensity)
-The third device has measured for three times: BBa_J23115 + BBa_E0240 in pSB1C3 backbone
The Mean: 1,299.34
The Standard Deviation: 155.48466
Protocols:
Digestion:
Plasmid DNA 10 microliter
Distilled water 6 ml
Buffer 2 ml
Enzyme 1 1 ml
Enzyme 2 2 ml
Put them into 1 ml Eppendorf and wait for 1 hour in the incubator set to 37 C.
Ligation:
Buffer 1 ml
Ligase 1 ml
Insert 5 ml
Vector 3 ml
Put them into 1 ml Eppendorf and wait for 1 hour in the incubator set to 37 C.
Transformation
1- Throw competent cell on ice
2- Mix ligation product (50 ml cell + 10 ml plasmid)
3- Incubate on ice for 30 min
4- Heat shock at 42C for 45 seconds
5- Incubate on ice for 5 min
6- Add 400 ml LB
7- Incubate at shaker set to 37 C 200 rpm for 2 hours
8- Centrifuge cells for 10 min at 3000 rpm
9- Discard supernatant and resuspend the pellet
10- Cultivate 25 ml to LB agar plates with antibiotic resistance and for positive control to nonantibiotic plates.
Overnight Culture:
1. Put 5 ml LB into a tube
2. Pick a single colony with loop from transformed plates
3. Put it into LB medium
4. Place the tube into shaker set to 37 C 200 rpm for 16 hours.
Flow Cytometer:
BD Accurium TM C6 Flow Cytometer.
By already fluorescently labeled compensation. Standard beads. The device is back flushed before every measurement.
Standard Flow Cytometer Protocol is applied.
In Flow Cytometer, SSC-H and FSC-H, FL1-H and FL4-H, Q1-UL, Q1-UR, Q1-LL, Q1-LR coordinate system plot type is used for determining whether to include or exclude. For outlier determining the samples are compared with the average and with the negative control samples.
As negative control we have used LB with E. coli (DH5a) samples and only LB samples.
Green Fluorescence is measured.
There is no practical limits on the number or rate of measurements taken with Flow Cytometer and the standard protocol. It is limitless. One can determine its limit however he/she wants. For example, if the requirements are appropriate for counting 50,000 cells this number becomes the limit. It can also be used for counting without arranging any limit. However, we have used the limit 20,000 cells to count one by one. So we actually could “measure cell-to-cell variation for the three required devices.” Which is an extra-credit requirement. By this way we could “measure single cells.”
Fluorescence is measured.
Units:
Molecules, MFI (Mean Fluorescence Intensity), % of E. coli (DH5a) expressing GFP
Second is used for measuring the time that 20,000 numbers of cells are counted by flow cytometer by measuring single cells depending to cell-to-cell variation.
Precision:
The range is basically counting only single cells to counting 100,000 single cells. In our measurement, 10,000 cells were acquired for flow cytometer and quadrant markers were applied using the negative control sample set (LB+E. coli). % GFP+Cells quadrant determining.
The significant figures for our measurement can be explained by an example: A MFI that is measured for the first set is as 1,194.19
Yes, the precision is the same across at the entire range.
By the guideline of the flow cytometer and by the results that the flow cytometer gave.
Accuracy:
It is calibrated in August 21, 2014 by the manufacturer.
It is calibrated by standard beads and by already fluorescently labeled compensation
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