Team:TU Eindhoven/Measurements





Interlab - Introduction



What is Interlab?



All competing iGEM teams were invited and encouraged to participate in the Second International InterLab Measurement Study in synthetic biology. The goal of the InterLab Study is to obtain fluorescence data of three specific devices constructed by iGEM teams. Last year 45 teams representing 18 countries from all around the world participated and delivered their fluorescence data of the three devices. The fluorescence data will be compared with the data from other iGEM teams and should result in a set of comparable data that could provide great insight into the variability of standard measurements. For more information about interlab, take a look at their website.
Figure 1: Worldmap showing the participating teams of 2014.


The devices



During this year’s InterLab study three devices are produced. Each of the plasmids contains a chloramphenicol resistance, a promotor (with unknown strength) and a GFP-gen built in behind it.



The three devices that have been constructed for this year are:
Figure 2: Showing the plasmid of device 1

To be able to compare all the three devices a positive and negative control are needed. Two other devices have been designed to serve as a positive and negative control.
For the positive and negative control two other devices are tested, namely:
  • Positive control: a GFP expression device (BBa_I2027) from the iGEM distribution kit
  • Negative control:
    • Cells without any plasmid
    • Empty vector (BBa_R0040) from the iGEM distribution kit



Methods



Producing devices 1,2 and 3, the positive and negative control



To produce the requested parts, traditional cloning methods have been used. The parts J23101 , J23106 and J23117 with pSB1C3 backbone and I13504 with pSB1A2 backbone were obtained from the cloning kit. All of the plasmids were amplified.
For amplification the plasmids were transformed into novablue. After transformation the cells were placed into a small culture to amplify the plasmids.
After amplification the plasmids were purified from the bacteria.
To purify the plasmids from the bacteria the Qiagen Miniprep Kit has been used. For more detailed information, take a look at Protocol 1: Plasmid amplification


To insert GFP obtained from part I13504 into the pSB1C3-plasmid of parts J23101, J23106 and J23117 restriction enzymes were used. SpeI and PstI-HF were used to make sticky ends in the pSB1C3 plasmid and XbaI and PstI-HF were used to make compatible sticky ends around the GFP-gen in pSB1A2.
For more detailed information, take a look at Protocol 2: Digestion.

Construct J23101 , J23106 and J23117 were purified from the other digestion product using PCR Purification.
For more detailled information, take a look at Protocol 3: PCR Purification.
The GFP-gen was purified from the rest of the pSB1A2-plasmid using an agarose gel.
For more detailled information, take a look at Protocol 4: Gel Purification.
The digested vectors (J23101, J23106 and J23117) and inserts I13504 were combined in a PCR-reaction using T4 Ligase.
For more detailled information, take a look at Protocol 5: Ligation.
Figure 3: An overview of the digestion and ligation method. The plasmids are digested by several restriction enzymes and the parts are ligated.

The plasmids produced in the ligation reaction (device 1, 2 and 3) and the positive (I2027) and negative (R0040) control obtained from the cloning kit, were again transformed into Nova Blue cells for amplification. The plasmids were obtained from the bacteria using Qiagen Miniprep Kit.
. For more detailled information, take a look at protocol 1: Plasmid Amplification.
After colony picking the transformed plasmids were checked for the correct length using Colony PCR.
For more detailled information, take a look at protocol 6: Colony PCR. .
To make sure the correct devices were obtained after plasmid amplification, the produced plasmids were sequenced.


Measuring GFP-expression



For protein expression the plasmids were transformed into BL21 (DE3) cells. Each of the plasmids had a constitutive promotor and hence no protein expression inducer was needed.
For more information, take a look at protocol 7: Protein Expression.
Once small-cultures were grown, fluorescence measurement could be performed. For measuring the amount of fluorescence we used the Tecan Infinite F500 platereader. Prior to loading the bacteria-culture in the plate the OD of the bacterial culture solution was adjusted to 0.5 by adding LB (pH 7.2). A blank measurement of LB (pH 7.2) was performed to correct for background signal.
For more information, take a look at protocol 8: Protein Expression Measurement.
Figure 4: The arrangement of the 96 black well
plate used for fluorescence measurement.
  • Row A: LB medium
  • Row B: Colony 1, 2, 3 of construct 1
  • Row C: Colony 1, 2, 3 of construct 2
  • Row D: Colony 1, 2, 3 of construct 3
  • Row E: Colony 1, 2, 3 of construct 4: negative control - empty BL21 bacteria
  • Row F: Colony 1, 2, 3 of construct 5: positive control - GFP with working promotor
  • Row G: Colony 1, 2, 3 of construct 6: negative control - BL21 without GFP and with B0062 promotor



Protocols

  1. Plasmid Amplification
  2. Digestion
  3. PCR Purification
  4. Gel Purification
  5. Ligation
  6. Colony PCR
  7. Protein Expression
  8. Protein Expression Measurement
  9. Agar Plates
  10. Antibiotic Stock Solutions



Results



Sequencing results


  • Device 1
    red:
    the promotor
    green:
    GFP

    TAT CTG ACT GAT CGC TTT TCA ATT CGG CGT TCT CAC GAG GCA GAA ATT TTC AGA ATT AAA AAA AAT CCT TAG CTT TCG CTA AGG ATG ATT TCT GGA ATT CGC GGC CGC TTC TAG AGT TTA CAG CTA GCT CAG TCC TAG GTA TTA TGC TAG CTA CTA GAG AAA GAG GAG AAA TAC TAG ATG CGT AAA GGA GAA GAA CTT TTC ACT GGA GTT GTC CCA ATT CTT GTT GAA TTA GAT GGT GAT GTT AAT GGG CAC AAA TTT TCT GTC AGT GGA GAG GGT GAA GGT GAT GCA ACA TAC GGA AAA CTT ACC CTT AAA TTT ATT TGC ACT ACT GGA AAA CTA CCT GTT CCA TGG CCA ACA CTT GTC ACT ACT TTC GGT TAT GGT GTT CAA TGC TTT GCG AGA TAC CCA GAT CAT ATG AAA CAG CAT GAC TTT TTC AAG AGT GCC ATG CCC GAA GGT TAT GTA CAG GAA AGA ACT ATA TTT TTC AAA GAT GAC GGG AAC TAC AAG ACA CGT GCT GAA GTC AAG TTT GAA GGT GAT ACC CTT GTT AAT AGA ATC GAG TTA AAA GGT ATT GAT TTT AAA GAA GAT GGA AAC ATT CTT GGA CAC AAA TTG GAA TAC AAC TAT AAC TCA CAC AAT GTA TAC ATC ATG GCA GAC AAA CAA AAG AAT GGA ATC AAA GTT AAC TTC AAA ATT AGA CAC AAC ATT GAA GAT GGA AGC GTT CAA CTA GCA GAC CAT TAT CAA CAA AAT ACT CCA ATT GGC GAT GGC CCT GTC CTT TTA CCA GAC AAC CAT TAC CTG TCC ACA CAA TCT GCC CTT TCG AAA GAT CCC AAC GAA AAG AGA GAC CAC ATG GTC CTT CTT GAG TTT GTA ACA GCT GCT GGG ATT ACA CAT GGC ATG GAT GAA CTA TAC AAA TAA TAA TAC TAG AGC CAG GCA TCA AAT AAA ACG AAA GGC TCA GTC GAA AGA CTG GGC CTT TCG TTT TAT CTG TTG TTT GTC GGT GAA CGC TCT CTA CTA GAG TCA CAC TGG CTC ACC TTC GGG TGG GCC TTT CTG CGT TTA TAT ACT AGT AGC GGC CGC TGC AGT CCG GGC AAA AAG GGC AAG GTG TCA CCA CCC TGC TCT TTT TCT TTA AAA CCG AAA AGA TTA CTT CGC GTT ATG CAG CTT CCT CGC TCA CTG ACT CGC TGC GCT CGG TCG TCG CTG CGC GAG CGT ATC AGC TCA CTC AAA GCG TAT ACG TAT CAC AGA TCA GGA TAC GCA AGG AAG AAC ATG TGA GCA AAG CAG CAA GCA AGG ATC GTA AAA GCC GCG GTG CTG CTT TCC AAG ATC GCT CTG ACA GAC ATC AAA AAT CGA GCC TCA GTC CAA GTT GGC GAA TCC GAC AGG GA
  • Device 2
    red:
    the promotor
    green:
    GFP

    TAC GAC TAC TAT AAA TAG GCG TAT CAC GAG GCA GAA TTT CAG ATA AAA AAA ATC CTT AGC TTT CGC TAA GGA TGA TTT CTG GAA TTC GCG GCC GCT TCT AGA GTT TAC GGC TAG CTC AGT CCT AGG TAT AGT GCT AGC TAC TAG AGA AAG AGG AGA AAT ACT AGA TGC GTA AAG GAG AAG AAC TTT TCA CTG GAG TTG TCC CAA TTC TTG TTG AAT TAG ATG GTG ATG TTA ATG GGC ACA AAT TTT CTG TCA GTG GAG AGG GTG AAG GTG ATG CAA CAT ACG GAA AAC TTA CCC TTA AAT TTA TTT GCA CTA CTG GAA AAC TAC CTG TTC CAT GGC CAA CAC TTG TCA CTA CTT TCG GTT ATG GTG TTC AAT GCT TTG CGA GAT ACC CAG ATC ATA TGA AAC AGC ATG ACT TTT TCA AGA GTG CCA TGC CCG AAG GTT ATG TAC AGG AAA GAA CTA TAT TTT TCA AAG ATG ACG GGA ACT ACA AGA CAC GTG CTG AAG TCA AGT TTG AAG GTG ATA CCC TTG TTA ATA GAA TCG AGT TAA AAG GTA TTG ATT TTA AAG AAG ATG GAA ACA TTC TTG GAC ACA AAT TGG AAT ACA ACT ATA ACT CAC ACA ATG TAT ACA TCA TGG CAG ACA AAC AAA AGA ATG GAA TCA AAG TTA ACT TCA AAA TTA GAC ACA ACA TTG AAG ATG GAA GCG TTC AAC TAG CAG ACC ATT ATC AAC AAA ATA CTC CAA TTG GCG ATG GCC CTG TCC TTT TAC CAG ACA ACC ATT ACC TGT CCA CAC AAT CTG CCC TTT CGA AAG ATC CCA ACG AAA AGA GAG ACC ACA TGG TCC TTC TTG AGT TTG TAA CAG CTG CTG GGA TTA CAC ATG GCA TGG ATG AAC TAT ACA AAT AAT AAT ACT AGA GCC AGG CAT CAA ATA AAA CGA AAG GCT CAG TCG AAA GAC TGG GCC TTT CGT TTT ATC TGT TGT TTG TCG GTG AAC GCT CTC TAC TAG AGT CAC ACT GGC TCA CCT TCG GGT GGG CCT TTC TGC GTT TAT ATA CTA GTA GCG GCC GCT GCA GTC CGG GCA AAA AAG GGC AAG GTG TCA CCA CCC TGC CCT TTT TTC TTT AAA ACC GAA AAG ATA CTT CGC GTA TGC AGC TTC TCG CTC ACT GAC TCG CTG CGC TCG GTC GTC GGC TGC GGC GAG CGT ATC AGC TCA CCT CAA GCG TAA TAC GTA TCC ACA GAA TCA GGA TAA CGC AGA AAA GAA CAT GTG AGC AAA GCT AGC CAA GTC AGA TCG GTT AAA GCC CGA TGC CTG GCG ATT TTC CAA GGA CTC CGC TCC TTG ACA GGC ATA CGA AAT CGA GCC TAG TCA GTG CAA TCC TGA ACG GAA C
  • Device 3
    red:
    the promotor
    green:
    GFP

    ..A ATG GCT TTA CTA TAA ATA GGC GTA TCA CGA GGC AGA ATT TCA GAT AAA AAA AAT CCT TAG CTT TCG CTA AGG ATG ATT TCT GGA ATT CGC GGC CGC TTC TAG AGT TGA CAG CTA GCT CAG TCC TAG GGA TTG TGC TAG CTA CTA GAG AAA GAG GAG AAA TAC TAG ATG CGT AAA GGA GAA GAA CTT TTC ACT GGA GTT GTC CCA ATT CTT GTT GAA TTA GAT GGT GAT GTT AAT GGG CAC AAA TTT TCT GTC AGT GGA GAG GGT GAA GGT GAT GCA ACA TAC GGA AAA CTT ACC CTT AAA TTT ATT TGC ACT ACT GGA AAA CTA CCT GTT CCA TGG CCA ACA CTT GTC ACT ACT TTC GGT TAT GGT GTT CAA TGC TTT GCG AGA TAC CCA GAT CAT ATG AAA CAG CAT GAC TTT TTC AAG AGT GCC ATG CCC GAA GGT TAT GTA CAG GAA AGA ACT ATA TTT TTC AAA GAT GAC GGG AAC TAC AAG ACA CGT GCT GAA GTC AAG TTT GAA GGT GAT ACC CTT GTT AAT AGA ATC GAG TTA AAA GGT ATT GAT TTT AAA GAA GAT GGA AAC ATT CTT GGA CAC AAA TTG GAA TAC AAC TAT AAC TCA CAC AAT GTA TAC ATC ATG GCA GAC AAA CAA AAG AAT GGA ATC AAA GTT AAC TTC AAA ATT AGA CAC AAC ATT GAA GAT GGA AGC GTT CAA CTA GCA GAC CAT TAT CAA CAA AAT ACT CCA ATT GGC GAT GGC CCT GTC CTT TTA CCA GAC AAC CAT TAC CTG TCC ACA CAA TCT GCC CTT TCG AAA GAT CCC AAC GAA AAG AGA GAC CAC ATG GTC CTT CTT GAG TTT GTA ACA GCT GCT GGG ATT ACA CAT GGC ATG GAT GAA CTA TAC AAA TAA TAA TAC TAG AGC CAG GCA TCA AAT AAA ACG AAA GGC TCA GTC GAA AGA CTG GGC CTT TCG TTT TAT CTG TTG TTT GTC GGT GAA CGC TCT CTA CTA GAG TCA CAC TGG CTC ACC TTC GGG TGG GCC TTT CTG CGT TTA TAT ACT AGT AGC GGC CGC TGC AGT CGG GCA AAA AAG GGC AAG GTG TCA CCA CCC TGC CCT TTT TCT TTA AAA CCG AAA AGA TTA CTT CGC GTT ATG CAG CTT TCT CGC TCA CTG ACT CGC TGC GCT CGT CGT TCG CTG CGC GAG CGG TAT CAG CTC ACT CAA AGC GTA ATA CGT TAT CAC AGA ATC AGG GAT ACG CAG AAA AGA AAC ATG TGA GCA AAG CAG CGA CGT CAG GAC GTT AAA AGG CCG GAT GCC TGG GTT TCA CAG GCT CGC CTC CTT GAC TAG CAT ACA AAA TCG AGC TCA GTC AAG TTG ACC TAA ACC CGA CAG CT
  • Positive Control
    red:
    the promotor
    green:
    GFP

    .GC TTG CCT GAC GCT CGA TTA CTA TAA TAG GCG TAT CAC GAG GCA GAA TTT CAG ATA AAA AAA ATC CTT AGC TTT CGC TAA GGA TGA TTT CTG GAA TTC GCG GCC GCT TCT AGA GTT GAT GGC TAG CTC AGT CCT AGG TAC AAT GCT AGC TAC TAG AGT CAC ACA GGA AAG TAC TAG ATG CGT AAA GGA GAA GAA CTT TTC ACT GGA GTT GTC CCA ATT CTT GTT GAA TTA GAT GGT GAT GTT AAT GGG CAC AAA TTT TCT GTC AGT GGA GAG GGT GAA GGT GAT GCA ACA TAC GGA AAA CTT ACC CTT AAA TTT ATT TGC ACT ACT GGA AAA CTA CCT GTT CCA TGG CCA ACA CTT GTC ACT ACT TTC GGT TAT GGT GTT CAA TGC TTT GCG AGA TAC CCA GAT CAT ATG AAA CAG CAT GAC TTT TTC AAG AGT GCC ATG CCC GAA GGT TAT GTA CAG GAA AGA ACT ATA TTT TTC AAA GAT GAC GGG AAC TAC AAG ACA CGT GCT GAA GTC AAG TTT GAA GGT GAT ACC CTT GTT AAT AGA ATC GAG TTA AAA GGT ATT GAT TTT AAA GAA GAT GGA AAC ATT CTT GGA CAC AAA TTG GAA TAC AAC TAT AAC TCA CAC AAT GTA TAC ATC ATG GCA GAC AAA CAA AAG AAT GGA ATC AAA GTT AAC TTC AAA ATT AGA CAC AAC ATT GAA GAT GGA AGC GTT CAA CTA GCA GAC CAT TAT CAA CAA AAT ACT CCA ATT GGC GAT GGC CCT GTC CTT TTA CCA GAC AAC CAT TAC CTG TCC ACA CAA TCT GCC CTT TCG AAA GAT CCC AAA CGA AAA GAG AGA CCA CAT GGT CCT TCT TGA GTT TGT AAC AGC TGC TGG GAT TAC ACA TGG CAT GGA TGA ACT ATA CAA ATA ATA ATA CTA GAG CCA GGC ATC AAA TAA ACG AAA GGC TCA GTC GAA AGA CTG GGC CTT TTC GTT TTA ATC TGT TGT TGT CGT GAA CGC TTC TCT ACT AGA GTC ACC ACT GGC TCC ACT TCG GTG GGC CTT TCT GCG TTT ATA TAC TAG TAG CGG CCG CTT GCA GTC CGG CAA AAA GGG CAA GTG TTC ACA CCC CTG CCT TTT TCT TAA AAA CGG AAA AGG ATA CTT TCG CGT ATG CAA GCT TCT CTC GCT TCA ATG TAC TCG CAT G
  • Negative Control
    Blue:
    The part sequence

    ATC GAC TTA ACT ATA AAT AGG CGT ATC ACG AGG CAG AAT TTC AGA TAA AAA AAA TCC TTA GCT TTC GCT AAG GAT GAT TTC TGG AAT TCG CGG CCG CTT CTA GAG TCC CTA TCA GTG ATA GAG ATT GAC ATC CCT ATC AGT GAT AGA GAT ACT GAG CAC TAC TAG TAG CGG CCG CTG CAG TCC GGC AAA AAA GGG TAA GGT GTC ACC ACC CTG CCC TTT TTT TTT AAA ACC GAA AAT ATT ACT TTT TTT TAT GCA CGC TTC CTC GCT AAC TGA TTC GCT GCG CGC GAG TCC TCT ATT GGG GTG AGC GGG ATC AGC TGC TGT TAA GAA GAA AAT CGG CTA GGA TAG AAA AGG GGA TAT TAA TGT TAG ATT TTT CGT GCA AAT GGC TAA TAA TAT GAT AGG AAC CTA TAA AGG CAC TTT TGC GGG AGT GTT GTC AGT CAA TTG AAG AAA TAA ATT TCA TCC TTA GTT TCA CAC TAA GAT ATC ATC TTG TCA ATC CTG TTA TAA GTA AAG CGA GCG CAA TAG AAA CTT GTG AAA CTA CAC ATA TAT TTC ATT ATT AAT ATA CTG AAG TTG CCC TGA GGT GAC CAT CTT ATT GCT ATC GAC AAT AGC TGC AGT CTA TAT ACA TCG ATC ACA GTA GGA CAA CGG GTG TGC GGC CTG CTC TCA TGA ATC ATT CAA CAA GCA TGA ATG AGG GGA TCA CGG ATT TAA CAA TAT TCA CCA CCC ACC ATC ACT GTG ATT GAT TGA TTT TGA TCA GTC AAT CTA CTC AGC AAC GAT ATT AAC TAG ACT TT


Fluorescence measurements results



From the measurements at OD 0.5 it results that LB gives a background fluorescence of around 6700. The presence of the cell with empty plasmid gives an additional 200 units background fluorescence. To correct for this background signal, the average fluorescence of the cell with empty plasmid has been subtracted from the measured fluorescence in the devices and positive control.
  • Data with a correction for background fluorescence
    Device 1 Technical replicate A Technical replicate B Technical replicate C Technical mean Technical SD
    Biological replicate 1:
    J23101_1     		32970.5556 30806.5556 30083.5556 31286.88889 1226.575812
    Biological replicate 2:
    J23101_2     		37509.5556 35238.5556 34900.5556 35882.88889 1158.47438
    Biological replicate 3:
    J23101_3     		40153.5556 38793.5556 39729.5556 39558.88889 568.1815046
    Biological mean 36877.8889 34946.2222 34904.5556 Total mean:
    35576.2222
    Biological SD 2966.26862 3267.22475 3937.96403 Total SD:
    3536.63871


    Device 2 Technical replicate A Technical replicate B Technical replicate C Technical mean Technical SD
    Biological replicate 1:
    J23106_1     		11651.5556 10828.5556 10988.5556 11156.22222 356.2923269
    Biological replicate 2:
    J23106_2     		13696.5556 12373.5556 12696.5556 12922.22222 563.1911063
    Biological replicate 3:
    J23106_3     		12328.5556 12296.5556 12672.5556 12432.55556 170.2077162
    Biological mean 12558.8889 11832.8889 12119.2222 Total mean:
    12170.3333
    Biological SD 850.606189 710.866295 799.562102 Total SD:
    843.72544


    Device 3 Technical replicate A Technical replicate B Technical replicate C Technical mean Technical SD
    Biological replicate 1:
    J23117_1     		286.555556 367.555556 319.555556 324.5555556 33.2565783
    Biological replicate 2:
    J23117_2     		297.555556 264.555556 358.555556 306.8888889 38.93869826
    Biological replicate 3:
    J23117_3     		103.555556 261.555556 142.555556 169.2222222 67.20284386
    Biological mean 229.222222 297.888889 273.555556 Total mean:
    266.888889
    Biological SD 88.9731545 49.2769949 93.9893611 Total SD:
    84.8580514


  • Data of the negative control
    Empty Cell Technical replicate A Technical replicate B Technical replicate C Technical mean Technical SD
    Biological replicate 1 6730 6851 6875 6818.666667 63.45777249
    Biological replicate 2 6761 7175 6792 6909.33333 188.280524
    Biological replicate 3 6787 6869 6888 6848 43.82541
    Total mean:
    6858.6667    		 Total SD:
    123.3937


    Cell with empty plasmid Technical replicate A Technical replicate B Technical replicate C Technical mean Technical SD
    Biological replicate 1 6831 6868 6982 6893.666667 64.26161806
    Biological replicate 2 6921 6955 6884 6920 28.9942523
    Biological replicate 3 6792 6891 6917 6866.666667 53.85371
    Total mean:
    6893.4444    		 Total SD:
    55.65591


    LB
    (pH 7.2)    		 1 2 3 4 5 6 7 8 9 10 11 12
    6724 6786 6660 6776 6735 6803 6758 6472 6616 6659 6452 6687 Total mean:
    6677.33
  • Data of the positive control
    Positive control Technical replicate A Technical replicate B Technical replicate C Technical mean Technical SD
    Biological replicate 1:
    I20270_1    		 9143.55556 8343.55556 8845.55556 8777.555556 330.1191704
    Biological replicate 2:
    I20270_2    		 9135.55556 10354.5556 9180.55556 9556.888889 564.3346131
    Biological replicate 3:
    I20270_3    		 8148.55556 8250.55556 8443.55556 8280.888889 122.3283378
    Biological mean 8809.22222 8982.88889 8823.22222 Total mean:
    8871.77778
    Biological SD 467.173296 970.657624 301.293139 Total SD:
    650.594903


From the measured fluorescence it can be deduced that: device 1 (part J23101) is very strong, device 2 (part J23106) is of average strength and that device 3 (part J23117) is weak. Compared to the positive control (part I20270), device 1 is four times stronger, device 2 is one and a half times stronger and device 3 is 32 times weaker. Device 3 hardly seems to work at all, but compared to the cell with empty plasmid still it gives a fluorescent expression that is 300 units higher.
Figure 5: The results of the fluorescence measurement with an OD600 of 0.5. The measurement has been done with arbitrary units .