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Location assay

Introduction

The object of the assay is to test whether signaling antisense RNA will bind to its complementary sequence encompassing the rbs and prevent translation. The location sequence unit which codes the information of the cell number is carried by a virus remains which was expressed inside the E.coli, and transmitted to other E.coli, while the antisense RNA is transcribed constantly inside themselves. If irrelevant mRNA are transmitted by irrelevant virus remains, their antisense will prevent translation from their mRNA.

In this assay, we used pBAD promoter to drive transcription of the location sequence which will be transmitted by a virus remains in our system. The strength of the promoter depends on the concentration of arabinose. By adjusting the concentration of arabinose, we will mimic the concentration of RNA emitted by a population of virus. On the other hand, we used c-pro as the promoter that drives transcription of antisense RNA. c-pro is the strongest constitutive promoter we were able to find in the igem parts registry.

In assay I, we examined the relative strength of pBAD (with eight different concentrations of arabinose) to c-pro. There are two main object for this assay; to quantify the amount of RNA transcribed by pBAD and c-pro, and to control the amount of RNA transcribed by pBAD which depends on the concentration of arabinose.

By using the result of assay I, we can make a condition which the amount of location sequence unit is less than that of antisense RNA, and in assay II we observe whether our antisense RNA is able to prevent translation or not. We prepared two location sequence units (L2 and L4), and two antisense RNA sequences (L2’ and L4’). L2’ is complementary to L2 and L4’ is complementary to L4. There are 4 samples.

• a) J61002/ terminator(rev)-gfp(rev)-L2-pBAD(rev)-cpro-L2’-terminator
• b) J61002/ terminator(rev)-gfp(rev)-L2-pBAD(rev)-cpro-L4’-terminator
• c) J61002/ terminator(rev)-gfp(rev)-L4-pBAD(rev)-cpro-L2’-terminator
• d) J61002/ terminator(rev)-gfp(rev)-L4-pBAD(rev)-cpro-L4’-terminator

(The location sequence unit contains the ribosome binding site (rbs).)

In sample a) and d), the antisense will bind to the location sequence unit, so there should be no gfp expressed.

In sample b) and c), the antisense is not complementary to the location sequence unit, so gfp should be expressed.

Assay I

• To quantify the amount of RNA transcribed by pBAD and c-pro.
• To control the amount of RNA transcribed by pBAD which depends on the concentration of arabinose.

Day 1
Prepare E.coli that contains the following four constructs:

• pSB1A3/ pBAD-LS-rbs-gfp-terminator
• pSB1A3/ pBAD-terminator
• J61002/ cpro-rbs-gfp-terminator
• J61002/ cpro-terminator.

1. Transform each of the above constructed plasmids to JM109 competent cells.
2. Incubate these plates at 37 oC for about twelve hours.

Day 2

Culture the full-growth bacteria.

1. Make full-growth bacteria for eight samples (A to H).

• Sample A; pSB1A3/ pBAD-LS-rbs-gfp-terminator, add 1.0x10^-2 M arabinose solution
• Sample B; pSB1A3/ pBAD-LS-rbs-gfp-terminator, add 1.0x10^-3 M arabinose solution
• Sample C; pSB1A3/ pBAD-LS- rbs-gfp-terminator, add 1.0x10^-4 M arabinose solution
• Sample D; pSB1A3/ pBAD-LS- rbs-gfp-terminator, add 1.0x10^-5 M arabinose solution
• Sample E; pSB1A3/ pBAD-LS- rbs-gfp-terminator, add 1.0x10^-6 M arabinose solution
• Sample F; pSB1A3/ pBAD-LS-terminator
• Sample G; J61002/ cpro- rbs-gfp-terminator
• Sample H; J61002/ cpro-terminator

1. Add tip which picked up one colony from the plate of each sample in 15 mL of LB broth (with 100ug/ml ampicillin).

1. Incubate sample at 37 oC with constant shaking at 180 rpm for more than 12 hours (until the medium become the state of full growth).

Day 3
Measure OD600 and make samples for measurement of gfp fluorescence.

1. Prepare 1 ml full growth medium for all samples in 100ml LB broth.
2. Make diluted arabinose solutions for sample A to E.

• a) Add “L-(+)-Arabinose, minimum 99%” 1.5 g into 10ml arabinose to make master mix solution of 1.0x10^-2^(-2)M arabinose for sample A.
• b) Add 150 ul master mix solution to 1350 ul LB broth to make 1.0x10^-3M arabinose solution for sample B.
• c) Add 150 ul 1.0x10^-3M arabinose solution to 1350 ul LB broth to make 1.0x10^(-4) arabinose solution for sample C.
• d) Add 150 ul 1.0x10^-4M arabinose solution to 1350 ul LB broth to make 1.0x10^(-5) arabinose solution for sample D.
• e) Add 150 ul 1.0x10^-5M arabinose solution to 1350 ul LB broth to make 1.0x10^(-6) arabinose solution for sample E.

1. Incubate sample at 25 oC with constant shaking at 180 rpm.
2. Extract 300 ul from each sample and measure OD600 once every hour.
3. Add arabinose solution of each concentration to the medium when the OD600 approaches 2.0.
4. Extract 1ml from each sample, centrifuge the medium and discard the supernatant to make E.coli pellet. Preserve all samples in a -20 oC freezer.

Day 4
Measure gfp fluorescence

1. Add 100 ul 8M urea buffer into each pellet and make suspension solution.
2. Incubate sample solutions at room temperature for 30 minutes.
3. Sonicate the cells. (10 seconds, 10% power.)
4. Cool the samples on ice and cool it down.
5. Repeat steps 3 and 4.
6. Centrifuge the tube at 150rpm, 2 minutes, 4 oC.
7. Measure the fluorescence.

Result

The result of assay I

As you can see from the figure, the strongest fluorescence observed was sample G, and it is followed by sample A, B, C, D, E. In this assay, we used two different types of vectors for plasmids; J61002 and pSB1A3. Copy number of these plasmids are the same (100 ~ 200) and all the plasmids use the same rbs and gfp. So, we assume that the amount of transcribed RNA closely correlate with the amount of gfp expression. The more gfp expressed, the stronger fluorescence must be observed, so the order of the strength of promoter must be following;

1. c-pro
2. pBAD with 1.0x10^-2 M arabinose
3. pBAD with 1.0x10^-3 M arabinose
4. pBAD with 1.0x10^-4 M arabinose
5. pBAD with 1.0x10^-5 M arabinose
6. pBAD with 1.0x10^-6 M arabinose

the correlation between the strength of pBAD output and the concentration of arabinose

Put the final fluorescence of c-pro pellet examined as 100% fluorescence, the remaining five samples was 70.6%, 25.8%, 16.8%, 1.35%, and 0.11%. As you can see from the figure, the concentration of arabinose positively correlates with the strength of pBAD output.

In sample A, the total amount of protein expression is only that of sample G. As for sample D and sample E, the amount of expression was too small compared to c-pro. We are not sure how much the emission of mRNA by virus might be, so we decided to adapt 1.0x10(-2) M and 1.0x10(-4) M as the concentration of arabinose for the next assay II.

Assay II

Test whether location sequence works or not.

Day 1
Prepare E.coli that contains the following four constructs:

-J61002/ terminator(rev)-gfp(rev)-L2-pBAD(rev)-cpro-L2’-terminator

-J61002/ terminator(rev)-gfp(rev)-L2-pBAD(rev)-cpro-L4’-terminator

-J61002/ terminator(rev)-gfp(rev)-L4-pBAD(rev)-cpro-L2’-terminator

-J61002/ terminator(rev)-gfp(rev)-L4-pBAD(rev)-cpro-L2’-terminator

1.Transform each of the above constructed plasmids to JM109 competent cells.

2.Incubate these plates at 37 oC for about twelve hours.

Day 2
Culture the bacteria to full growth. 1. Make full growth for eight samples (A to H).

Sample A;J61002/ 4-gfp-L2-pBAD(rev)-cpro-L2’-4, add 1.0x10^(-2) M arabinose solution

Sample B;J61002/ 4-gfp-L2-pBAD(rev)-cpro-L4’-4, add 1.0x10^(-2) M arabinose solution

Sample C;J61002/ 4-gfp-L4-pBAD(rev)-cpro-L2’-4, add 1.0x10^(-2) M arabinose solution

Sample D;J61002/ 4-gfg-L4-pBAD(rev)-cpro-L4’-4, add 1.0x10^(-2) M arabinose solution

Sample E;J61002/ 4-gfp-L2-pBAD(rev)-cpro-L2’-4, add 1.0x10^(-4) M arabinose solution

Sample F;J61002/ 4-gfp-L2-pBAD(rev)-cpro-L4’-4, add 1.0x10^(-4) M arabinose solution

Sample G;J61002/ 4-gfp-L4-pBAD(rev)-cpro-L2’-4, add 1.0x10^(-4) M arabinose solution

Sample H;J61002/ 4-gfg-L4-pBAD(rev)-cpro-L4’-4, add 1.0x10^(-4) M arabinose solution

2. Add tip which picked up one colony from the plate of each sample in 15 mL of LB broth (with 100ug/ml ampicillin).

3. Incubate sample at 37 oC with constant shaking at 180 rpm for more than 12 hours (until the medium become the state of full growth).

Day 3
Measure Optical Density (600) and make pellets for measurement of gfp fluorescence.

1. Prepare 1 ml full growth medium for all samples in 100ml LB broth.

2. Incubate sample at 25 oC with constant shaking at 180 rpm.

3. Extract 300 ul from each sample and measure OD600 once every hour.

4. Add arabinose solution of each concentration to the medium when the OD600 approaches 2.0.

5. Extract 1ml from each sample, centrifuge the medium and discard the supernatant to make E.coli pellet. Preserve all samples in a -20 oC freezer.

Day 4
Measure Fluorescence

1. Add 100 ul 8M urea buffer into each pellet and make suspension solution.

2. Incubate sample solutions at room temperature for 30 minutes.

3. Sonicate the cells. (10 seconds, 10% power.)

4. Cool the samples on ice and cool it down.

5. Repeat steps 3 and 4.

6. Centrifuge the tube at 150rpm, 2 minutes, 4 oC.

7. Measure the fluorescence.

Result

The result of assay II

If antisense RNA which we designed was available, the gfp couldn’t be translated in sample A, D, E, H. So we expect fluorescence from sample B, C, F, G and no fluorescence from sample A, D, E, H.

However, as you can see from the figure, there was no fluorescence of gfp being observed.

In fact, we sequenced our sample after the assay. As the result, actually our sample A and sample E has been terminator(rev)-gfp(rev)-L2-pBAD(rev), sample B and sample F has been terminator(rev)-gfp(rev)-L2-pBAD(rev). It seems that we have failed in the process of ligation.

Several reasons could be considered for the result of the assay. -reverse version of pBAD did not work Because, we had the result which did not depend on the concentration of arabinose, there is possibility that pBAD promoter did not work well. If the promoter did not work, the transcription does not occur, so the fluorescence of gfp won’t be observed. However, sequence result of pBAD reverse had no problem.

-gfp expressed by our plasmid was unavailable If pBAD(rev) was proper, then the reverse version of gfp expressed must have some problem. Actually, sequence result of gfp(rev) had no problem. However, in our parts, rbs exists inside “location sequence”, so there are extra DNA array between rbs and gfp. There is possibility that this extra array became obstacle for gfp fluorescence.

To make sure whether the array did become an obstacle or not, we may do the next Assay III.

Assay III

Delete the DNA array between rbs and gfp by PCR. Use primers which anneal to gfp(rev) from 5’ to 3’ and rbs(rev) from 3’ to 5’. Phosphorylate the PCR product and ligate them, to make new plasmids bellow:

a) J16002/ terminator(rev)-gfp(rev)-L4(new)-pBAD(rev)-cpro-L4’-terminator

b) J16002/ terminator(rev)-gfp(rev)-L4(new)-pBAD(rev)-cpro-L2’-terminator

The specific array of L4(new) will be rbs-location sequence4, and the specific array of original L4 was location sequence4-rbs-locationsequence4.

We expect antisense RNA transcribed from plasmid a) works and block the translation of gfp and no enough fluorescence being observed. On the other hand, antisense RNA transcribed form plasmid b) isn’t complementary with location sequence unit, so translation won’t be blocked and gfp fluorescence will be observed.