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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:

Device 1: backbone pSB1C3 containing J23101 (promotor) and I13504 (GFP)
Device 2: backbone pSB1C3 containing J23106 (promotor) and I13504 (GFP)
Device 3: backbone pSB1C3 containing J23117 (promotor) and I13504 (GFP)

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 on 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

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 .

@@ Line 9: / Line 9: @@
 <ul>
 <li>See why we think we deserved a <a href="https://2015.igem.org/Team:TU_Eindhoven/Achievements">Golden Medal</a>.</li>
-<li>We joined and completed the <a href="https://2015.igem.org/Team:TU_Eindhoven/Project/Interlab">Interlab Study</a>.</li>
 </ul>
 </span>

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