Team:HokkaidoU Japan/Shuffling Kit/Examples

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Maestro E.coli

+

Maestro E. coli

-

Optimization Kit

+

Shuffling Kit

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Promoter Selector

Promoter Selector

Let's select the best promoter for Kanamycin resistance by Promoter Selector.

Let's select the best promoter for Kanamycin resistance by Promoter Selector.

-

For a demonstration we decided to optimize the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector.(fig.1)

+

For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).

-

If the concentration of Kanamycin was high, the colony with strong promoter will survive. Therefore, only one or two colors of colonies would appear. +

If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate. - If the concentration of Kanamycin was low, colonies with weak promoters will be able to survive. This way many colors of colonies would appear. (fig.2). + If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).

- + -
fig.1
+
fig.1 Different promoter express each colors.
- + -
fig.2
+
fig.2 Difference of Kanamycin concentration.

Method

Method

-

Optimum concentration of Kanamycin: in LB is 50 mg/ml +

Optimum concentration of Kanamycin: in LB is 50 mg/ml
- We prepared 4 different concentration plates. + We prepared optimum and other 3 concentration plates.

Line 49: Line 49: Vector: pSB1C3 Vector: pSB1C3

-

We cloned Kanamycin resistant gene from pSB1K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).

+

We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).

- Culture: 37 °C, for 24h + Culture: 37 °C, for 48h

Results

Results

-

We didn't have time to culture anymore. + - So we coudn't  see the diffference beween plates A to D. +

- However,we were able to see different colors on the plate. + - The colonies were too small to identify the color. +
fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.
- The picture below is plate B. +
-

+
-
fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.
+
fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.
+ +

After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZ&alpha expression system in Promoter Selector as negative control to estimate the success of BsaI digestion. We got 0-7 blue colonies which was expressing LacZ&alpha. +

+ +

We observed mixed colored colonies.  They  may have been transformed by two or more Promoter Selector. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies. +

+ + +

+ In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=1. +

+ +

Conclusion

Conclusion

-

Our Promoter Selector seemed to work. We got several colors colonies on Kanamycin plates. +

- This means that Kanamycin Resistance gene was successfully ligated. + Big difference did not appear among each Kanamaicin concentration. - We need more than 24 hrs to identify the colors. + + In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084505 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest.

+

+ The other reason of why , colonies derived from K1084405 had the largest number may be, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from assay of only once , higher concentration of Kanamycin and much number of trials than this time will be needed. +

+

+ From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled. However it was not adopted to all colonies. As a result of assembling, we succeeded in making colorful colonies appear on one plate. +

RBS Selector

RBS Selector

-

Let’s optimize two reporter genes and make various colors on one plate!

+

4 colors

- +

Let’s create all combinations by two reporter genes and make various colors on one plate!

The RBS Selector we made, can randomize the strength of RBSs in the operon. The RBS Selector we made, can randomize the strength of RBSs in the operon. - For a demonstration, we decided to optimize two genes; mRFP1 (BBa_E1010) and LacZα (BBa_I732006) (fig.4). LacZα makes the colony blue. mRFP1 makes the colony red. + For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZα (BBa_I732006) (fig.5). LacZα makes the colony blue. mRFP1 makes the colony red.

- - https://static.igem.org/mediawiki/2013/5/5d/ROK_demo_color_HokkaidoU_2013.png"> +
fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).
-
fig.4
+
-

When the RBS upstream of mRFP1 was strong and the RBS upstream was weak, the colony should be red. When the RBS upstream of mRFP1 was weak, and the RBS upstream was strong, the colony should be blue. So when if the strength of RBS upstream both genes were the same, colony will be purple.(fig.5)

+

When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).

- + -
fig.5
+
fig.6 Each combinations of RBS make different colors.
-

Method

+

Method

-
• Used GGA vector (BBa_K1084301) and tandem RBS (BBa_K1084302). and assembled with
• +
• Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).
• Spread X-GAL（250 mg）on LBC plate.
• Spread X-GAL（250 mg）on LBC plate.
• -
• Cultured for 37 °C, 26h.
• +
• Cultured at 37 °C for 26h.
-

Results

+
+ +
fig.7 The colonies showed red, blue, white, and purple.
+
+

Results

+

+ We got colorful colonies; red, blue, white, and purple. + + +

+
+ +

Conclusion

+

- We got many colored colonies,red, blue, deep blue, and purple. + We can say that our RBS Selector worked exactly!! + The RBSs upstream of 2 genes were randomly assembled and they had different expression level.

+ + +

64 colors

+ +

+ Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate! +

+ +

+ As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR,  One-pot Golden Gate Assembly had done for making 64 kinds of different constructs. +

+ + +
+ +
fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.
+
+ +

+

Method

+
+
• Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.
• +
• +
• Cultured at 37 °C, 48h.
• +
+

+
- + -
fig.6 The colonies showed red, blue, deep blue, and purple.
+
fig.9 The result plate of 64 pattern randomizing.
-

Conclusion

+

Results

+

+ + +

+ + Many yellowish green (amilGFP) and red color (mRFP1) colonies appeared, but these colonies are undesirable colonies.They did not contain inserts of all three color proteins. As a Golden Gate assembly result, green color (strong aeBlue and amilGFP expression) appeared, and the insert DNA length was confirmed that the insert DNA has same length of Golden Gate Assembly. + Other color colony, white, weak green, weak amilGFP expression colonies were observed. We did not have time to confirm  all inserts were ligated. +

+

+

Conclusion

- We can say that our RBS Selector worked!! - The RBSs uptsream 2 genes were randomized and they had many levels of expressions. +

+ mRFP1 expression might be caused by the remaining of mRFP1 expression DNA used as template for GGA VECTOR producing by PCR (K1084401). +

+ +

+ However, the blue and green colonies appeared on the plate had the insert DNA length same with ideal Golden Gate Assembling product. Our Shuffling kit have partially worked!

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Demonstrations for Usecase Example

We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!

Promoter Selector

Let's select the best promoter for Kanamycin resistance by Promoter Selector.

For this demonstration we decided to use the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).

If the concentration of Kanamycin was high, the colony with strong promoter would survive. Therefore, only one or two colors would appear and indicate the first and second biggest occupancy rate on the plate. If the concentration of Kanamycin was low, colonies with even weak promoters could be able to survive. So in this way many colors of colonies would appear (fig.2).

fig.1 Different promoter express each colors.
fig.2 Difference of Kanamycin concentration.

Method

Optimum concentration of Kanamycin: in LB is 50 mg/ml
We prepared optimum and other 3 concentration plates.

• Plate A: Kanamycin 125 mg per plate
• Plate B: Kanamycin 250 mg per plate
• Plate C: Kanamycin 500 mg per plate (optimum concentration)
• Plate D: Kanamycin 1000 mg per plate

Gene Vector: pSB1C3

We cloned Kanamycin resistance gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).

Culture: 37 °C, for 48h

Results

fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.
fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.

After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZ&alpha expression system in Promoter Selector as negative control to estimate the success of BsaI digestion. We got 0-7 blue colonies which was expressing LacZ&alpha.

We observed mixed colored colonies. They may have been transformed by two or more Promoter Selector. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.

In fig.4, legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table referrers the number of each Promoter Selector’s colony. These data are collected by n=1.

Conclusion

Big difference did not appear among each Kanamaicin concentration. In this experiment, number of colonies derived from K1084405 (containing K1084010 promoter) had the largest rate on each plate. This result suggests that the colonies which had K1084505 could cost little resource of transcription and translation for expression of Kanamycin resistance gene, and the rest of resource had been spared to cell growth. Thus, the number of colonies might be the largest.

The other reason of why , colonies derived from K1084405 had the largest number may be, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from assay of only once , higher concentration of Kanamycin and much number of trials than this time will be needed.

From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled. However it was not adopted to all colonies. As a result of assembling, we succeeded in making colorful colonies appear on one plate.

RBS Selector

4 colors

Let’s create all combinations by two reporter genes and make various colors on one plate!

The RBS Selector we made, can randomize the strength of RBSs in the operon. For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZα (BBa_I732006) (fig.5). LacZα makes the colony blue. mRFP1 makes the colony red.

fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).

When the RBS of mRFP1 was stronger than that of LacZα, the colony would be red. When in the opposite case, the colony would be blue. And when the strengths of both RBSs were same, colony would be white or purple (fig.6).

fig.6 Each combinations of RBS make different colors.

Method

• Assembled promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102).
• Spread X-GAL（250 mg）on LBC plate.
• Cultured at 37 °C for 26h.
fig.7 The colonies showed red, blue, white, and purple.

Results

We got colorful colonies; red, blue, white, and purple.

Conclusion

We can say that our RBS Selector worked exactly!! The RBSs upstream of 2 genes were randomly assembled and they had different expression level.

64 colors

Let’s create all combinations by three reporter gene and using tandem RBS, make various colors on one plate!

As the tandem RBS and the RBS selector demonstration, we randomized 64 patterns of RBS-CDS combination. By adding tandem RBS, three kinds of CDSs and GGA VACTOR, One-pot Golden Gate Assembly had done for making 64 kinds of different constructs.

fig.8 The 64 patterns of RBS-CDS combination constructed by RBS Selector.

Method

• Used tandem RBS (BBa_K1084302), GGA VECTOR that BsaI and overhang was added by PCR from K1084401, eforRed (BBa_K592012), aeBlue (BBa_K864401) and amilGFP (BBa_K592010) are assembled by Golden Gate Assembly.