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. The positive control contains GFP in a plasmid with a promotor of which is known it works. Two negative controls were constructed. The first control contains an empty vector and the second one consists of a completely empty bacteria. 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; 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 cutting 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.

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
  spoiler..
• Negative Control
  spoiler..

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.