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| K1319003 in pSB1C3 || G00100_Alternative || K1319003_R || 930 || ???
| K1319003 in pSB1C3 || G00100_Alternative || K1319003_R || 930 || ???
== 28th ==
= October =
= October =

Revision as of 16:55, 29 September 2014


In order to detect the pathogens fast, specifically and inexpensively we are building sensor cells to detect these pathogens. These sensor cells can identify pathogens in very low concentration by responsing to specific extracellular molecules either secreted by or displayed on the pathogens. These molecules trigger a fast fluorescence response by our immobilized sensor cells which will be measured by our device. Aachen Cellock standingup.png

More information will be available soon!



  • SOE-PCR step 2


  • we transformed some BioBricks and did a colony PCR
Aachen 14-05-23 COLONY-PCR.jpg
Colony PCR picture



  • preculture for competent NEB10β cells
  • submit samples for sequencing
  • make masterplates of transformed BioBricks
  • centrifuge and freeze overnight expression culture of J23101.E0240 and K516132


  • made competent NEB10β cells - several things went wrong.. we'll see (remember: pre-cool centrifuge, always check if it's indeed spinning, frequently check OD of the culture)
  • colony PCR on the transformed clones looked awful. There were too many cells in the 10 µl reaction volume. Some seals weren't fully closed. Basically only primers and smear except for the positive control which contained a plasmid template instead of cells
  • 2x 500 ml of LB and three sterile flasks have been made



  • plasmid preps
  • transformation of different reporter strains
    • NEB
      • pSEVE641_BsFbFP
      • pSEVA234_LasR
      • pSEVE641_BsFbFP pSEVA234_LasR
      • pSEVE641_BsFbFP pSB1C3_C0179
    • BL21
      • pSEVE641_BsFbFP pSEVA234_LasR
      • pSEVE641_BsFbFP pSB1C3_C0179


  • Philipp and Michael made 100x stocks for Hartmans media
  • Michael and Vera made about 25 HM+C-plates without Glucose
  • K731520 iLOV was plated on a HM+C plate


  • Michael made 25 new HM+C plates with 1 % agar and 4 g/L glucose
  • He also added 170 µl of the Glucose stock (500 g/L) to the Glucose-free HM+C-plates
  • 1 L of sterile HM+Glucose was prepared
  • K731520 iLOV was plated on HM+C+Glucose and HM+C+Glucose drops, 3x 5 ml HM+C+Glucose precultures were inoculated (17:00)


  • K731520 iLOV did not grow, neither on the plates, nor on in the liquid media. The most probable cause is that the E. coli is missing some vitamins
  • based upon the M9 recipes by the Knight and Endy labs on OpenWetWare, we made a 20 ml supplement stock solution that can be added to 1x HM media
Component supplement for 1x HM [g/L] final 1x concentration
Casamino acids 2 2 g/L
Thiamine hydrochloride 0.3 300 mg/L
MgSO4/MgSO4*7H2O 0.242 / 0,494 2 mmol/L
CaCl2 0.011 0.1 mmol/L

The powders for 1 L of 1x HM were dissolved in 20 ml of a 10 % w/v Casamino acid stock solution and sterile-filtered (.22 µm PES). To make agar chips, we can add 1 ml to the hot agar mixture, together with the three 500 µl 100x stocks for the HM.

We made 250 ml of HM+C+Glucose+Supplements plates with 1.5 % agar.

Then we plated the following combinations:

Construct HM+C+Glucose+Supplements LB+C
K731520 iLOV - +
J23101.E0240 + +

We also prepared a 150 ml HM+C+Glucose+Supplements culture with K731520 iLOV to hopefully make agar chips on Friday.

6x of last weeks J23115.E0240 clones were plated on LB+C and 5 ml LB+C precultures were inoculated for plasmid preparation.


The K731520 iLOV strain did not grow on the HM plate, while another strain with the J23101.E0240 construct did. Both have the pSB1C3 backbone. To confirm the plasmids and inserts, Michael set up a colony PCR:

ID Template Product Length Result
1 J23101.E0240 #5 1233
2 J23101.E0240 #6 1233
3 J23101.E0240 #5 plasmid 1233
4 K731520 iLOV LB colony 2053
5 K731520 iLOV plasmid 2053
6 K731520 GFP plasmid 2437
7 water none

Reaction volume per tube is 15 µl, GoTaq Green Mastermix and the VF2 and VR primers. You can find the durations and temperatures are in this table:

parameter duration temp [°C]

Florian mini-prepped the 6 overnight cultures of J23115.E0240 clones. He used 3 ml instead of 1.5 and eluted twice with 25 µl nuclease-free water. Everything else was according to the protocol of the illustra plasmidPrep Mini-Spin Kit.

The resulting DNA concentrations are listed in this table:

clone # concentration [ng/µl]
1 73.5
2 94.5
3 100.5
4 53
5 82
6 138

In the evening, we plated some strains on the different media to investigate why the K731520 iLOV did not grow on the HM+suppl. plate. (Note: in the morning, Michael re-inoculated the HM+C+suppl. shake flask with cells from the LB-plate and by afternoon the culture did grow to a high cell density.)

Construct Host strain LB+C HM+C+Glucose HM+C+Glucose+supplements
J23101.E0240 NEB10beta ++ - ++
K731520 iLOV DH5alpha ++ - (+)
K131026 NEB10beta ++ - -
K131026 DH5alpha ++ - +

Philipp inoculated J23101.E0240 in LB and HM+C+Glucose+supplements



  • ...



  • Stefan and Vera made electrocompetent E.coli.
  • Arne prepared cultures for Saturday, 14-8-2 of Renés iLOV and K131026


  • Testing the OD measurement device and comparing it to the Spektrophotometer and the Platereader.
  • Also testing K131026 and K731520 iLOV for fluorescence in the Platereader.
  • Heat shock Transformation into NEB TOP 10 cells of I746909.
  • Electroshock transformation of pET17-Gal3 into E.coli rosetta.


  • OD Measurements of the iGEM device in comparison to the Spektrometer.
  • Preparing culutures for cryos of K131026 and K731520 iLOV


  • Arne and Michael made cryo-stocks of K731520 iLOV and K131026 in NEB/BL21/DH5alpha, I746909 in BL21 and pET17-His-SNAP-YFP-Gal3 in E. coli Rosetta (DE3)
  • Michael plasmid-prepped most of them using 1.5 ml culture and eluted with 1x 50 µl of ddH2O.
combination concentration [ng/µl]
I746909 BL21 #1 73.5
I746909 BL21 #2 45
I746909 BL21 #3 49
K731520 iLOV DH5alpha 60
K131026 DH5alpha 150
pET17-Gal3 #1 30.5
pET17-Gal3 #2 6.4
pET17-Gal3 #3 6.3
pET17-Gal3 #4 9.4
pET17-Gal3 #5 10.1
pET17-Gal3 #6 8.2
pET17-Gal3 #7 13.8
pET17-Gal3 #8 6.9
pET17-Gal3 #9 10.2

To confirm the quality of pET17-Gal3-transformations, the purified plasmids were test-digested:

14-08-04 Test-Digest.png
Test digest
clones were test-digested
combination cut products[bp]
I746909 BL21 #1 2029, 947
K731520 iLOV DH5alpha 2029, 1780
K131026 DH5alpha 2029, 1848
pET17-Gal3 #1 3086, 923, 1262

All pET17-Gal3 clones were positive and clone #1 was selected for further experiments.


  • Stefan, Arne and Michael assembled a VR=2.5 L bioreactor for cultivation of a 1 L expression culture
  • Two precultures of 20 ml LB+A were inoculated at 19:00
  • Anna transformed K746909 into BL21 cells and K1319000 into NEB10beta


  • Eshani and Arne transformed J04450 in pSB1K3 and pSB1A3 in NEB Beta cells
  • They also did a plasmid prep of J04450 in pSB1C3 and Flo's vectors
  • Arne made precultures of NEB Beta and DH5 alpha cells
  • Arne and Michael inoculated the fermenter at 11:40, induced the fermentation of pET17-Gal3. The fermentation is expected to run 24 h


  • Plasmid restriction of I20260 (EcoRI,PstI), J23115 (EcoRI, SpeI), K516032 (XbaI,PstI), and J23101 (EcoRI, SpeI)


  • Ligation of J23115 and K516032 to J23115.K516032 and J23101 and K516032 to J23101.K516032
  • Plasmid prep of I20260, K516032 and B0034
  • Restriction of Plasmid I20260, K516032, B0034 with EcoRI and PstI
  • Gel with restricted I20260, K516032 and B0034
  • purification of vector backbone, pSB1A2, pSB3K3 and pSB1C3
  • Restriciton of snythesized TEV-Protease with EcoRI and PstI


  • Ligation of J23101.K516032 into pSB3K3 and J23115.K516032 into pSB3K3 and K1319004 into pSB1C3
  • Transformation of K1319004 in pUC and pSB1C3 as well as J04450 in pSB1K3 and pSB1A3



Michael, Vera and Eshani prepared 50 ml LB+antibiotic overnight-cultures of pSBX-vectors from Heidelberg.


  • In the morning, at 10:15, Anna inoculated the precultures for the interlab study experiment.
  • Michael prepared cryo-stocks of the pSBX-carrying E. coli from the overnight cultures. He also purified each pSBX-vector, eluting with 15+30 µl water:
vector concentration [ng/µl]
pSBX1A3 111
pSBX4A5 14.1
pSBX1C3 31
pSB4C5 98.5
pSBX1K3 18
pSBX4K5 30
pSBX1T3 39
constitutive expression plasmid 73

  • Anna did PCRs for Gibson-Assembly of K1319003 into pET17. Duplicates of 25 µl reaction volume (12.5 µl Q5 2x Master Mix, 2x 1.25 µl primers, 2 µl template)
PCR tube # components
1 and 2 pET17 + pET17_Gal3_Gib_F + pET17_Gal3_Gib_R
3 and 4 K1319003 + K1319003_Gib_F + K1319003_Gib_R

The PCR conditions:

step temperature [°C] duration
denature 98 30", 98 °C for 10", 55 °C for 30", 72 °C for 2'15"
denature 98 10"
anneal 50 (insert) 55 (backbone) 30"
elongate 72 0'30" (insert) 2'15" (backbone)
elongate 72 2"
store 8 indefinite

Finally, Florian did the Gibson assembly and heat shock transformation into NEB10beta cells.

At 10:15 Arne inoculated the primary cultures of the Interlab Study experiment and began with regular fluorescence measurements.


  • Anna made masterplates of yesterdays transformed cells.


  • Anna made precultures of 3 clones from each prepared master palte and inoculated precultures for OD/F measurements as well as chip production on the 7th.


  • Anna made cryos of from the precultures and Michael and Arne purified the plasmids:
plasmid strain resistance vector # of clone picked concentration [ng/µl]
K1319000 in I20260 NEB10ß K pSB3K3 1
K1319000 in I20260 NEB10ß K pSB3K3 3
K1319000 in I20260 NEB10ß K pSB3K3 5
K1319001 in I20260 NEB10ß K pSB3K3 1
K1319001 in I20260 NEB10ß K pSB3K3 5
K1319001 in I20260 NEB10ß K pSB3K3 6
K1319002 in I20260 NEB10ß K pSB3K3 1
K1319002 in I20260 NEB10ß K pSB3K3 5
K1319002 in I20260 NEB10ß K pSB3K3 6
K1319001_GFP Fusion in I20260 NEB10ß K pSB3K3 4
K1319001_GFP Fusion in I20260 NEB10ß K pSB3K3 5
K1319001_GFP Fusion in I20260 NEB10ß K pSB3K3 6
K1319002_GFP Fusion in I20260 NEB10ß K pSB3K3 3
K1319002_GFP Fusion in I20260 NEB10ß K pSB3K3 4
K1319002_GFP Fusion in I20260 NEB10ß K pSB3K3 5
His-SNAP-YFP-K1319003 NEB10ß A pET17 3
His-SNAP-YFP-K1319003 NEB10ß A pET17 4
His-SNAP-YFP-K1319003 NEB10ß A pET17 6
  • Elution was performed with 2 * 15 µl of nuclease free water.


Arne and Michael were able to analyze the sequencing data from the clones of GFP_Reach 1, GFP_Reach 2 and K1319008.

GFP_Reach 2 clone number 3 and 5 were fine. With the included Leu to Ile mutation. GFP_Reach 1 clone number 4 and 5 were fine and did not contain the Leu to Ile mutation. Clone 6 was fine but contained the Leu to Ile mutation from the Reach 1 quick change mutations.

Further we will use the GFP_Reach 1 clone 4 and the GFP_Reach 2 clone 4.

Transformation of GFP_Reach 1 clone 3 and GFP_Reach 2 clones number 3 and 5 were performed together with the TEV-Protease to create two plasmid construct.

The GFP_Reach 1 and GFP_Reach 2 constructs were also restricted and ligated into the pSB1C3 vector from the pSB3K3 vector.


Vera made master plates for the transformation from the day before.

Also PCRs were made from pSBXA3, I20260 and K131900 for a Gibson Assembly. The PCRs were checked with a gel electrophoresis.




Rene, Arne and Philipp made cultures of K1319013, K1319013 + K1319008, K1319013 + K1319008 + iPTG, K1319014, K1319014 + K1319008, K1319014 + K1319008 + iPTG, B0015 (negatove control) and I20260 (positive control). iPTG was added at an OD of ~0,5. Inoculation was done via precultures in 500 ml shake flasks (50 ml filling volume). Media was always LB. Cultivation was done at 37 degrees Celsius and 300 rpm. Starting OD was aimed to be 0,1. Inoculation occured directly from the precultures. Samples will be taken every hour and checked for OD and fluorescence (Spektrometer and Platereader respectively).

Vera did a pasmid preparation from the cultures of the day before which were K1319013, K1319013 + K1319008, K1319013 + K1319008 + iPTG, K1319014, K1319014 + K1319008, K1319014 + K1319008 + iPTG, B0015 (negatove control) and I20260 (positive control). The plasmid will then be cut with EcoRI and PstI and the results will be put on an agarose gel in order to perform a restriction test. Also plasmids of K1319013 and K1319014 will be cut with EcoRi and SpeI. K1319008 will be cut with XbaI and PstI. These will then be ligated together and then ligated into a pSB1A3 vector via the 3A assembly (vector cut with EcoRI and PstI). These constructs will be transformed into BL21 (and NEB as a backup). The created construts will be known as K1319018 (K1319013.K1319008) and K1319019 (K1319014.K1319008).

Florian made precultures of the master plates from the day before (K1319008, K1319013, K1319015 and pSBX1A3 with Gal3).

Arne also inoculated 4 cultures for the further testing of the OD/F device (the F part). The cultures are 2 shake flasks of I20260 and 2 shake flasks of B0015.

Furthermore Nina did a growth experiment with DH5alpha for the Neanderlab school experiment. It is done at 37 degrees Celsius and 300 rpm in TB Media. It was inoculated for an OD of 1,5. Samples are taken every 30 minutes and tested for OD (Spektrometer).

Anna also made chips with K1319013 + K1319008, K1319014 + K1319008, K1319013, K1319014, B0015 and K131026. These will be inocubated for an hour at 37 degress Celsius. Then they will be induced with iPTG/HSL and fotos will be made every 30 minutes to check for fluorescence (GFP).

Florian tested our OD/F device with a dilution test. Samples were checked with Spektrometer (OD) our OD/F device (fluorescence) and platereader (fluorescence).

Anna and Vera made SDS Gels.

Stefan inoculated a culture of K1319008, B0015 as well as I20260 to check wether the results from our construct are from a wrongly done Gibson Assembly with a still functioning superfolder GFP.



  • Michael did a check PCR on several cryo cultures. All samples with G00100_Alternative+K1319004_check_R combinations resulted in a strong band at ~2300 bp that we cannot explain. All G00100_Alternative+K1319004_check_R combinations resulted in a strong band at 900 bp that we cannot explain either. We concluded that the annealing temperatures were wrong and favored unspecific products. Therefore we decided to do a gradient PCR to find out the optimal annealing temperatures for our new primers.

Gradient PCR to test new primers

Florian and Michael did gradient PCR with these new primers:

name sequence

Three PCRs with different primer combinations were run. In all of them the templates were K1319004 in pSB1C3, K1319008 in pSB1C3 and I746909 in pSB1C3.

The first gradient PCR tested the G00100_Alternative + G00101_Alternative combination:

primer_F primer_R template expected length best annealing temperature
G00100_Alternative G00101_Alternative K1319004 in pSB1C3 1057  ???
G00100_Alternative G00101_Alternative K1319008 in pSB1C3 1245  ???
G00100_Alternative G00101_Alternative I746909 in pSB1C3 1221  ???
G00100_Alternative G00101_Alternative water ---  ???
Aachen 14-09-26 gradientPCR 1.png
Gradient PCR 1
the primers were G00100_Alternative and G00101_Alternative and they worked well at all temperatures from 55-65 °C.

The second gradient PCR tested the G00100_Alternative + I746916_check_R combination:

primer_F primer_R template expected length best annealing temperature
G00100_Alternative I746916_check_R K1319004 in pSB1C3 none  ???
G00100_Alternative I746916_check_R K1319008 in pSB1C3 none  ???
G00100_Alternative I746916_check_R I746909 in pSB1C3 820  ???
G00100_Alternative I746916_check_R water ---  ???
Aachen 14-09-26 gradientPCR 2.png
Gradient PCR 2
the primers were G00100_Alternative and I746916_check_R and they worked well at all temperatures from 55-65 °C. Apparently the K1319008 template contained I746916.

The third gradient PCR tested the G00100_Alternative + K1319004_check_R combination:

primer_F primer_R template expected length best annealing temperature
G00100_Alternative K1319004_check_R K1319004 in pSB1C3 541  ???
G00100_Alternative K1319004_check_R K1319008 in pSB1C3 502  ???
G00100_Alternative K1319004_check_R I746909 in pSB1C3 none  ???
G00100_Alternative K1319004_check_R water ---  ???
Aachen 14-09-26 gradientPCR 3.png
Gradient PCR 3
The primers were G00100_Alternative and K1319004_check_R and they worked well at all temperatures from 60-68.1 °C. To our disappointment, the K1319008 template did not contain K1319004. It is unclear why the 5 bands of K1319008 and I746916 look different.

The results of these three PCRs are:

  1. The KAPA2G Fast ReadyMix worked well
  2. all three primers work well at >65 °C annealing temperature
  3. the K1319008 template was contained I746916 instead of the intended K1319004 ORF

It was concluded that a similar check PCR with 65 °C annealing temperature will be done on all plasmids and cryos of K1319008.


First Michael transformed K1319001, K1319002, K1319003 and K1319004 (all in pSB1C3) into NEB10beta cells. He tested the PCR machine for semi-automated heat-shocking by splitting the 50 µl cells with the plasmid into 2x 25 µl. All 100 µl were plated for all construct/machine combinations.

Vera transformed several constructs into chemically competent BL21(DE3) cells.

Philipp and Michael did colonyPCR on all plasmids, cryos and colonies that should contain the K1319004 sequence.

Vera also made check a PCR on galectin-constructs:

label primer_F primer_R expected length result
Gal3 in pSBX1A3 #1 G00100_Alternative K1319003_R 1684  ???
Gal3 in pSBX1A3 #2 G00100_Alternative K1319003_R 1684  ???
Gal3 in pSBX1A3 #3 G00100_Alternative K1319003_R 1684  ???
Gal3 YFP #3 pETGal3_seq_F K1319003_R 867  ???
Gal3 YFP #3 pETGal3_seq_F K1319003_R 867  ???
Gal3 YFP pet17 AmpR pETGal3_seq_F K1319003_R 867 or none  ???
pET17 Gal3 #1 pETGal3_seq_F K1319003_R none  ???
K1319003 in pSB1C3 G00100_Alternative K1319003_R 930  ???